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JNK activation of BIM promotes hepatic oxidative stress, steatosis and insulin resistance in obesity.
Sara A. Litwak1, Lokman Pang1,2, Sandra Galic1, Mariana Igoillo-Esteve3, William J. Stanley1,2, Jean-Valery Turatsinze3, Kim Loh1, Helen E. Thomas1,2, Arpeeta Sharma4, Eric Trepo5,6, Christophe Moreno5,6, Daniel J. Gough7,8, Decio L. Eizirik3, Judy B. de Haan4, Esteban N. Gurzov1,2,a
1St Vincent’s Institute of Medical Research, Melbourne, Australia. 2Department of Medicine, St. Vincent’s Hospital, The University of Melbourne, Melbourne, Australia. 3ULB Center for Diabetes Research, Université Libre de Bruxelles (ULB), Brussels, Belgium. 4Oxidative Stress Laboratory, Basic Science Division, Baker Heart and Diabetes Institute, Melbourne, VIC, Australia. 5CUB Hôpital Erasme, Université Libre de Bruxelles (ULB), Belgium. 6Laboratory of experimental Gastroenterology, Université Libre de Bruxelles (ULB), Belgium. 7Hudson Institute of Medical Research, Clayton, VIC, Australia. 8Department of Molecular and Translational Science, Monash University, Clayton, Vic, Australia.
aPresent address, to where correspondence and reprint requests should be addressed:
Dr Esteban N. Gurzov ULB Center for Diabetes Research Université Libre de Bruxelles Campus Erasme, Route de Lennik 808, B 1070 Brussels Belgium Phone: +32 2 5556242 Fax: +32 2 5556239
Email: [email protected]
Disclosure statement: The authors declare no conflict of interest
Running Title: BIM regulates lipid metabolism in hepatocytes.
1
Diabetes Publish Ahead of Print, published online September 19, 2017 Diabetes Page 2 of 62
ABSTRACT
The BCL 2 family are crucial regulators of the mitochondrial pathway of apoptosis in normal physiology and disease. Besides their role in cell death, BCL 2 proteins have been implicated in the regulation of mitochondrial oxidative phosphorylation and cellular metabolism. It remains unclear, however, whether these proteins have a physiological role in glucose homeostasis and metabolism in vivo. Here we report that fat accumulation in the liver increases JNK dependent BIM expression in hepatocytes. To determine the consequences of hepatic BIM deficiency in diet induced obesity, we generated liver specific BIM knockout (BLKO) mice. BLKO mice had lower hepatic lipid content, increased insulin signalling and improved global glucose metabolism. Consistent with these findings, lipogenic and lipid uptake genes were downregulated and lipid oxidation enhanced in obese BLKO mice.
Mechanistically, BIM deficiency improved mitochondrial function and decreased oxidative stress, oxidation of protein tyrosine phosphatases and ameliorated activation of PPAR γ/SREBP1/CD36 in hepatocytes from high fat fed mice. Importantly, short term knockdown of BIM rescued obese mice from insulin resistance, evidenced by reduced fat accumulation and improved insulin sensitivity. Our data indicate that BIM is an important regulator of liver dysfunction in obesity, and a novel therapeutic target for restoring hepatocyte function.
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INTRODUCTION
Obesity is a major risk factor for the development of severe complications such as non
alcoholic fatty liver disease (NAFLD), cardiovascular disease and diabetes (1). The
liver has an essential role in the regulation of glucose homeostasis, producing glucose
during fasting periods to prevent hypoglycaemia, maintaining brain function and
survival. In obese individuals, excessive nutrients are stored in the liver as fat droplets
(hepatosteatosis), which can eventually result in inflammation, insulin resistance and
diabetes (2; 3). Obesity triggers activation of the c Jun N terminal kinase (JNK)
pathway in hepatocytes with subsequent insulin resistance as well as steatosis (4; 5).
The mechanisms of obesity induced JNK activation include endoplasmic reticulum
(ER) and oxidative stress, Toll like receptors (TLRs) and inflammatory cytokines, such
as tumor necrosis factor (TNF) α (6). Hepatocyte‐specific deletion of both JNK1 and
JNK2 improved glucose and insulin tolerance, increased hepatic insulin action and
lowered fasting blood glucose levels in obese mice (7). However, the molecular
pathways and proteins mediating JNK induced hepatocyte dysfunction remain to be
fully elucidated.
The BCL 2 proteins are key regulators of apoptotic pathways (8), but growing
evidence indicates that these proteins can also play an important role in glucose
homeostasis and metabolism (9; 10). We have recently reported that loss of the BCL
2 protein PUMA influences circulating leptin levels and food intake in mice (11).
Moreover, BCL 2 associated death promoter (BAD) phosphorylation activates
glucokinase and thus controls hepatic gluconeogenesis and insulin secretion in
pancreatic β cells (12; 13). In addition, BCL 2 proteins have been reported to
stimulate glucose consumption (14) and regulate glucose metabolism through
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mitochondrial activity (15) or Ca2+ trafficking (16; 17). This dual role in metabolism/apoptosis of BCL 2 proteins is reminiscent of the action of cytochrome c, a protein that functions as a crucial component for oxidative phosphorylation in the mitochondria, but when displaced to the cytosol induces cleavage of caspases and cell death (9).
BCL 2 interacting mediator of cell death (BIM) is a BCL 2 homology 3 (BH3) only protein widely expressed in tissues (18). Saturated free fatty acids trigger cell death in hepatocytes in culture through BIM up regulation (19 21). Fat accumulation and hepatocellular carcinoma in liver specific STAT5 knockout mice is associated with down regulation of BIM and PUMA (22). In line with these findings, liver apoptosis in BCL XL or MCL 1 knockout mice is ameliorated by simultaneous BIM deletion
(23). While it is well accepted that BIM has a role in apoptosis induction in hepatocytes, its role in the regulation of hepatic lipid and glucose metabolism in vivo is unclear. Moreover, it remains unknown whether dysregulation of BIM may contribute to the development of metabolic diseases such as liver steatosis and insulin resistance. Here, we show that BIM is induced in hepatocytes during obesity. To clarify the role of BIM, we generated a novel liver specific BIM deficient mouse model and observed that BIM deletion decreases fat accumulation, and improves insulin signalling in vivo. These novel observations highlight BIM as a key mediator of liver dysfunction in obesity.
RESEARCH DESIGN AND METHODS
Human samples.
We studied 19 biopsy specimens of patients undergoing a liver biopsy for medical
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reasons in our institution. The clinical characteristics of these patients are shown in
Supplementary Figure 1E. The biopsies were collected after approval of the Erasme
Hospital Ethics Committee. Written informed consent was obtained from each
participant.
Mice.
Mice were maintained at St. Vincent’s Institute animal care facility on a 12h light
dark cycle in a temperature controlled room and obtained food and water ad libitum.
Bimlox/lox mice were generated on a C57BL/6 background as previously described
(24). Tissue specific deletion of BIM was generated by crossing Bimlox/lox mice with
Alb Cre (C57BL/6) mice (JAX). Male mice were kept on regular chow (20% protein,
6% fat and 3.2% crude fibre) or placed at 8 10 weeks of age on a high fat diet (SF04
027 Speciality Feeds, Perth, Western Australia) for 14 20 weeks. The nutritional
composition of the high fat diet was 18.4% protein, 23.5% fat and 4.7% crude fibre.
The calculated composition of fatty acids in the high fat diet is: 14.31% total saturated
fats, 7.54% total mono unsaturated fats, and 2.07% total polyunsaturated fats. In this
diet, 46% of total energy is from lipids, 20% of total calculated energy from protein
and the remainder from carbohydrates. All animal studies were conducted at St
Vincent’s Institute (Melbourne, Australia) following the guidelines of the Institutional
Animal Ethics Committee.
Metabolic analysis.
After 14 15 weeks on the diet and 24h of acclimatisation, oxygen consumption,
energy expenditure, respiratory exchange ratio, activity and food intake were assessed
using a Comprehensive Lab Animal Monitoring System (CLAMS, Columbus
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Instruments, OH). Data was averaged for 2 dark and light cycles. Body and tissue composition were determined by MRI scanning (EchoMRI, Houston, TX). After 18
20 weeks of chow/high fat feeding, mice were sacrificed and tissues were weighed, and collected for further analysis. Serum triacylglycerides (Wako Diagnostic,
Richmond, VA), insulin (Merck Millipore, Billerica, MA), and β hydroxybutyrate
(Sigma Aldrich, Darmstadt, Germany) concentrations were determined using commercial kits and following manufacturer’s instructions.
Mice were fasted for 4h before performing intra peritoneal insulin tolerance test.
Insulin (Actrapid, Novo Nordisk, Bagsværd, Denmark), at a dose of 0.65mU/g, was injected intraperitoneally and blood glucose was measured after tail bleeding. Mice were fasted for 6h before an intra peritoneal glucose (2mg/g, Baxter, Deerfield, IL) or pyruvate (2mg/g, Sigma Aldrich) tolerance test.
Cell culture and treatments.
Mouse hepatocytes were isolated by a two step collagenase A (0.05% w/v; Roche
Diagnostics) perfusion method as described previously (25). Where indicated cells were treated with 0.5mM sodium palmitate in the presence of 1% w/v fatty acid free
BSA (26). The JNK inhibitor SP600125 (Santa Cruz Biotechnology, CA) was used at
10 M (27). JNKs (JNK1 and JNK2) were knocked down transiently in primary hepatocytes using JNK specific siRNAs (Cell Signaling #6232) as described (25); scramble siRNAs were used as a control.
Adenovirus production and viral transduction.
Adenoviruses carrying green fluorescent protein (GFP) as reporter and BIM shRNA or scrambled control were developed by Vector Biolabs (Malvern, PA). The 21bp
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targeted sequence of BIM was GACGAGTTCAACGAAACTTAC. Virus amplification,
purification, titration and verification were done by Vector Biolabs. Adenoviruses
with CMV (Cytomegalovirus) promoter driving the expression of Cre recombinase
protein fused to the enhanced GFP (EGFP) were developed by the University of Iowa
Viral Vector Core Facility (Iowa City, IA). Adenoviral transduction of hepatocyte
cultures was carried out 24h after plating at a viral dose of 2x106 plaque forming unit
(pfu)/ml. For in vivo treatment, 19 week old lox/lox or 20 week high fat fed (HFF)
C57BL/6 mice were intravenously injected with a viral dose of 1x109pfu in 200 L of
saline.
Real-time PCR, RNA sequencing and data analysis.
Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, The
Netherlands). RNA quality analysis, library preparation and sequencing were
performed by the Australian Genome Research Facility (AGRF, Melbourne,
Australia). The sequencing was performed on an Illumina HiSeq2500 using v4
chemistry. An average of 21,6 million single end read of 100 nucleotides long were
obtained per sample. The raw data generated during the sequencing procedure
(Supplementary Table 1) was deposited in the Gene Expression Omnibus database
(GEO), access number GSE102982. The lists of up/downregulated genes/transcripts
and association with canonical pathways was determined using Ingenuity Pathway
Analysis (IPA) (Supplementary Table 2).
Real time PCR was performed using the Rotor Gene RG 3000 machine (Corbett
Research; Qiagen, Hilden, Germany) and the TaqMan PCR Master Mix (AmpliTaq
Gold with GeneAmp kit; Applied Biosystems). Analyses were performed with the
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ddCT method using 18s as internal controls. Probe details are provided in
Supplementary Table 3.
Western blot.
Muscle, liver and white adipose tissue (gonadal) were lysed using RIPA buffer and total proteins were extracted and resolved by SDS PAGE, transferred onto a nitrocellulose membrane and immunoblotted with primary antibodies
(Supplementary Table 4). The intensity values for the protein bands were corrected by the values of the housekeeping protein β actin or α tubulin used as loading controls. Total (reversible and irreversible) PTP oxidation was assessed as previously described (25).
Biochemistry analysis.
Primary mouse hepatocytes were isolated from lox/lox and BLKO mice, and analysed on a Seahorse XF24 Flux Analyzer (Seahorse Bioscience, North Billerica, MA) according to a previously described protocol (28). Calculations of parameters of mitochondrial respiratory function (28) included subtraction of non mitochondrial respiration from all mitochondrial respiration parameters. The data was normalised to total protein content/well measured by BCA assay (Thermo Fisher Scientific,
Waltham, MA) according to manufacturer's instructions.
ROS determinations.
ROS was measured in lox/lox and BLKO hepatocytes using MitoSox Red (1.2 M,
Invitrogen) and 2’,7’–dichlorofluorescin diacetate (0.2 M, DCFDA, Abcam), according to manufacturer’s instructions.
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Immunofluorescence and histology.
Hepatocytes and paraffin sections were fixed with paraformaldehyde (4% cells, 2.5%
sections) and permeabilized with triton X100 (0.3% cells, 0.1% sections). The fixed
cells were incubated overnight at 4°C with the following primary antibodies: rabbit
anti BIM (1/200, Cell Signaling) and mouse anti cytochrome c (1/200, BD
Biosciences, San Jose, CA). The secondary antibody used for visualization was either
fluorescein isothiocyanate (FITC) or Rhodamine conjugated antibodies developed
against rabbit or mouse (Jackson ImmunoResearch, Westgrove, PA EUA), diluted at
1/200. Nuclei were counterstained with Hoechst 33342 (HO, 10 g/ml; Sigma
Aldrich) before mounting.
For histological analysis tissue fixed in formalin, and sections (5µm) were stained
according to standard procedures (25). Guinea pig anti insulin antibody (Dako,
Glostrup, Denmark) was detected with Alexa Fluor®555 conjugated goat anti guinea
pig IgG antibody (Life Technologies, Carlsbad, CA). Mouse anti glucagon antibody
(Sigma Aldrich) was detected with Alexa Fluor®647 conjugated goat anti mouse IgG
antibody (Life Technologies). Nuclei were stained with DAPI reagent (Life
Technologies). Images were analysed on a Nikon A1R A1 confocal microscope
(Nikon Corporation, Tokyo, Japan). The percentage of insulin and glucagon positive
staining per islet was analysed using ImageJ software.
Hepatic lipid content was analysed on frozen sections of lox/lox and BLKO livers by
Oil Red O staining following a previously published protocol (29). Slides were
imaged using an Aperio digital slide scanner (Leica, Wetzlar, Germany).
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TdT mediated dUTP X nick end labelling (TUNEL) in paraffin sections was determined using In Situ Cell Death Detection kit, TMR red (Sigma Aldrich).
Liver paraffin sections were dewaxed, antigen retrieval was performed and endogenous peroxidases were neutralized with 0.3% hydrogen peroxide. Liver sections were then blocked in 1% bovine serum albumin and incubated with anti nitrotyrosine antibody (Merck Millipore) overnight at 4°C. Secondary antibody, biotinylated anti rabbit immunoglobulin 1:500 (Dako) was then added for 10 min, followed by horseradish peroxidase–conjugated streptavidin, diluted 1:500 (Dako), and incubated for 30 min in 3,3′ diaminobenzidine tetrahydrochloride (Sigma
Aldrich) with hematoxylin counterstain. Images were visualized under light microscopy (at 20x) and quantitated using Image Pro Plus.
Palmitate oxidation assay.
Measurement of palmitate oxidation capacity of hepatocytes using [14C] palmitate has been described previously (30). The radioactivity of the culture medium as well as the acid soluble intermediates after chemical extraction of cellular lipids was measured.
14 Total oxidation was then calculated as a function of both [ C] CO2 produced and incomplete oxidation products.
Statistical Analysis.
Statistical significance was determined by a two tailed paired Student’s t-test or
ANOVA with Bonferroni correction. P values < 0.05 were considered statistically significant.
RESULTS
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Obesity induces BIM expression in hepatocytes through JNK activation.
As a first step towards elucidating the role of BCL 2 proteins in the regulation of
glucose and lipid metabolism in the liver, we determined whether obesity affects the
expression of hepatic BCL 2 proteins in age matched C57BL/6 mice fed either a high
fat diet (23.5% fat; 46% energy from fat) or standard chow diet for 14 weeks.
Consistent with the development of obesity, HFF mice have increased white adipose
tissue and liver weights and become glucose intolerant and insulin resistant when
compared with chow fed lean controls (Supplementary Figs. 1A-D). To assess the
BCL 2 protein profile, livers from HFF obese and chow fed lean mice were harvested
and homogenised for Western blot analysis with BCL 2 antibodies. We noted a
striking increase in the expression of BIM and MCL 1 and decrease of one splicing
isoform of BAX in the livers from HFF mice (Fig. 1A). The role of MCL 1 in liver
dysfunction in obesity has been previously studied (31) and we thus focused on BIM.
Hepatocytes constitute approximately 80% of the liver volume. Hence, we next
determined whether the observed increased BIM expression could be ascribed to
changes specifically in these cells. Primary hepatocytes were freshly isolated from
chow and HFF mice, and we confirmed increased BIM protein levels in cells from
obese mice (Fig. 1B). Importantly, we observed a trend to increased BIM protein
expression in liver biopsies from patients with different degrees of NAFLD when
compared to non tumorigenic healthy livers (Fig. 1C, Supplementary Fig. 1E).
To determine if the modulation of BCL 2 proteins is due to a direct effect of saturated
fats in the liver, we isolated hepatocytes and exposed them to the saturated free fatty
acid palmitate. BIM, but not MCL 1/BAX spliced, was differentially up regulated in
palmitate treated cells (Fig. 1D, Supplementary Fig. 2A). The unsaturated free fatty
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acid oleate, which antagonizes the deleterious metabolic effects of palmitate (32), did not affect BIM expression (Supplementary Fig. 2B). Therefore, we hypothesized that saturated fat accumulation in the liver triggers BIM up regulation in the hepatocytes. It has been described that BIM transcriptional expression in hepatocytes is regulated by FoxO3a and JNK/c Jun (20; 21; 33), but we did not observe activation of FoxO3a in the livers of HFF mice or in palmitate treated hepatocytes
(Supplementary Figs. 2C-D). On the other hand, JNK and its downstream transcription factor c Jun were phosphorylated/activated in fatty livers (Fig. 1E).
Moreover, palmitate treated hepatocytes induced JNK/c Jun phosphorylation and transcriptional upregulation of BIM (Figs. 1F-G). Inhibition of JNK by SP600125 or by specific siRNAs prevented palmitate induced BIM expression (Figs. 1H-I). These results suggest that saturated fat increases BIM expression in a JNK/c Jun dependent fashion, and raise the possibility that the deleterious effects of obesity can be mediated, at least in part, via differential modulation of BCL 2 proteins.
A new mouse model with hepatocyte-specific deletion of BIM: the BLKO mice.
To ascertain whether the activation of hepatic BIM contributes to liver steatosis and obesity, we deleted BIM specifically in hepatocytes. For this purpose, BIMlox/lox
(lox/lox) mice were bred with Alb Cre mice to generate liver specific BIM knockout
(BLKO) mice on a C57BL/6 background (Supplementary Fig. 3A). BLKO mice appeared normal and did not exhibit differences in body weight (Supplementary Fig.
3B). However, the liver specific BIM deficient mice have slightly less adiposity, as assessed by monitoring body composition by magnetic resonance imaging (MRI) and by measuring epididymal fat pad weights (Figs. 2A-B). Total liver weight was similar when comparing BLKO and lox/lox mice, but hepatic fat accumulation tended to be
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decreased in the BLKO (Fig. 2C). In keeping with their reduced adiposity, BLKO
mice exhibited improved insulin and pyruvate tolerance (Figs. 2D-E). The observed
reduction of adiposity was accompanied by a trend for increased oxygen consumption
and increased energy expenditure (Supplementary Fig. 3C). No differences were
observed regarding respiratory exchange ratio, ambulatory activity or daily food
intake (Supplementary Fig. 3C).
Obese BLKO mice have improved insulin sensitivity and reduced liver steatosis.
To assess the impact of hepatic BIM deficiency on the development of obesity, 8 10
week old male lox/lox and BLKO mice were fed a high fat diet for 14 weeks and their
body weight and metabolic status assessed (Fig. 3A). HFF BLKO mice gained similar
weight as their lox/lox littermates and there were no differences in global adiposity or
fat pad weights (Figs. 3B-C). Interestingly, liver total weight was lower in HFF
BLKO mice (Fig. 3C). The reduced liver weight was associated with decreased
steatosis, as assessed by gross morphology, histology (haematoxylin and eosin
staining), Oil Red O staining (lipid droplets) and by measuring the percentage of
hepatic fat by MRI (Figs. 3D-F). HFF BLKO mice also exhibited decreased fasting
blood triacylglyceride levels (Fig. 3G).
Steatosis is a key contributor to obesity induced defects in insulin receptor (IR)
signalling cascade (34). We observed that obese BLKO mice respond better to
glucose, insulin and pyruvate tolerance tests as compared to control lox/lox mice
(Figs. 3H-J). Insulin induced IR phosphorylation in the liver was not different
between obese BLKO and lox/lox controls, but the interaction between IRS1 and the
p85 regulatory subunit of PI3K and the induction of p AKT were significantly
enhanced in obese BLKO mice (Fig. 3K, Supplementary Fig. 4A). As expected,
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insulin induced AKT phosphorylation was not altered in muscle or white adipose tissue (Supplementary Figs. 4B-C), confirming a liver specific phenotype.
Inactivation of the insulin pathway in the liver induces compensatory pancreatic β cell mass expansion and increased insulin secretion (34; 35), suggesting that the increased insulin sensitivity present in the liver of BLKO mice could induce the opposite phenotype. Consistent with this, we observed reduced levels or circulating insulin and insulin staining in islets from HFF BLKO mice (Figs. 3L-M). The improved glucose homeostasis in HFF BLKO mice was accompanied by enhanced energy expenditure and oxygen consumption (Supplementary Fig. 4D). No differences were observed in respiratory exchange ratio, food intake and ambulatory capacity (Supplementary Fig.
4D). BIM is associated with apoptosis induction in hepatocytes. Thus, we measured apoptosis by TUNEL analysis (Supplementary Fig. 4E), inflammatory markers by qPCR in the liver (Supplementary Fig. 4F), and serum alanine aminotransferase
(ALT) levels (Supplementary Fig. 4G). There were no differences in all these parameters between obese BLKO and lox/lox mice. Taken together, these results demonstrate that BIM deficiency in the liver reduces steatosis and improves insulin sensitivity in obesity, independently of its effects on apoptosis.
BIM deficiency decreases oxidative stress and improves fatty acid metabolism in the liver.
In order to gain mechanistic insight on the direct role of BIM activity in hepatocytes, we run a series of in vitro experiments, starting with RNA seq analysis of three preparations of freshly isolated BLKO and lox/lox control hepatocytes
(Supplementary Fig. 5A, Supplementary Table 1). BIM deficiency led to major changes in the metabolic gene network of the cells (Supplementary Fig. 5B,
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Supplementary Table 2). Interestingly, there was modulation of both mitochondrial
related and oxidative phosphorylation genes (Supplementary Fig. 5C). In line with
these findings, the maximal respiratory capacity and ATP production were higher in
BIM deficient hepatocytes as determined by mitochondrial oxygen consumption
(OCR) using Seahorse bioanalysis (Supplementary Fig. 5D). Moreover, confocal
microscopy demonstrated that BIM co localizes with cytochrome c at the
mitochondria in liver samples from obese C57BL/6 mice in vivo (Fig. 4A), and in
primary hepatocytes after palmitate treatment in vitro (Fig. 4B). BCL 2 proteins have
been associated to mitochondrial structure and production of reactive oxygen species
(ROS) (15; 36; 37). Indeed, decreased mitochondrial function and enhanced ROS
were observed in neuroblastoma cells conditionally expressing BIM (38). We found
that saturated free fatty acids induced mitochondrial ROS production in lox/lox
hepatocytes but not in BIM deficient cells, as measured by two different assays,
namely mitochondrial specific MitoSOX Red and 2’,7’–dichlorofluorescin diacetate
(DCFDA) (Figs. 5A-B). The chronic generation of ROS and consequent oxidative
stress observed in fatty livers of obese individuals is at least in part the result of
deficient mitochondrial function mediated by enhanced uptake and oxidation of
energy substrates (39). Protein tyrosine phosphatases (PTPs) are targets for ROS in
the liver through oxidation of their cysteine residue in the catalytic site (25; 40).
Consistent with our in vitro observations, livers from HFF BLKO mice have
decreased oxidative stress as indirectly assessed by nitrotyrosine levels, PTPs
oxidation and tyrosine phosphorylated proteins (Figs. 5C-D). STAT1 and JNK
activation, both dependent of ROS production (40; 41), were also decreased or tended
to decrease in obese BLKO livers respectively (Fig. 5E).
Steatosis is accompanied by ROS induced expression of the transcription factors
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peroxisome proliferator activated receptor (PPAR) γ and the downstream sterol regulatory element binding protein (SREBP)1, which induce the expression of the lipogenic enzymes Stearoyl CoA desaturase (SCD)1 and Acetyl CoA carboxylase
(AAC)1 and suppress lipid oxidation (42). PPAR γ also induces the expression of the fatty acid transporter CD36/FAT, which promotes the uptake of free fatty acids in the liver (43; 44). Interestingly, PPAR γ expression is regulated by c Jun (and hence JNK activity) during steatosis (45). PPAR γ and SREBP1 were reduced in the livers of
HFF BLKO mice (Figs. 5F-G). Consistent with this result, the expression of liver
SCD1 and AAC1 was lower in obese BLKO than in control mice, as was the expression of CD36 (Fig. 5G). Moreover, we observed down regulation of CD36 in our RNA seq analysis of BLKO and lox/lox hepatocytes (Supplementary Tables 1-
2, Supplementary Fig. 5C), and confirmed this result by qPCR in control and palmitate treated cells (Supplementary Fig. 5E). On the other hand, lipid oxidation, as assessed by indirect calorimetry and serum levels of β hydroxybutyrate, was increased in HFF BLKO mice (Figs. 5H-I). Taken together, these results indicate that the deletion of BIM in the liver prevents the deleterious effects of high fat diet induced obesity and insulin resistance, as well as hepatic lipogenesis, decreased lipid oxidation and steatosis.
Partial BIM inactivation prevents diet-induced insulin resistance in vivo.
To test if partial deletion of BIM has also a protective effect against the development of liver steatosis and insulin resistance, we generated male mice with heterozygous hepatic BIM deficiency (approx. 50% BIM reduction; Fig. 6A) and challenged them with a high fat diet. Interestingly, even partial reduction of hepatic BIM was sufficient to improve insulin sensitivity in obesity (Figs. 6B-C). One concern regarding our
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genetically modified models is that we cannot exclude compensatory effects by
developmental recombination of BIM, contributing to the observed phenotype. To
address this caveat, 19 week old lox/lox mice were inoculated with 1x109pfu of either
adenovirus AdCRE (expressing the CRE recombinase) or AdControl, and insulin
tolerance tested 4 weeks later. In line with the data obtained in BLKO mice, lox/lox
adult mice with AdCRE mediated BIM deletion showed an improved insulin
tolerance (Fig. 6D-E).
Finally, we developed an adenovirus expressing GFP as reporter with a shRNA to
selectively knockdown BIM in adult mice (Fig. 6F), and assessed the potential
translational relevance of our findings. The adenovirus infected 100% of hepatocytes
in culture, with a MOI of 2x106 pfu/ml achieving more than 90% knockdown of BIM
(Fig. 6F). Hepatocytes with BIM knockdown had a greater capacity to oxidise
palmitate than control cells (Fig. 6G). Next, mice were placed on a high fat diet for 20
weeks and insulin resistance was confirmed by insulin tolerance tests with a chow
lean group control. Obese mice were divided in two groups, with no differences in
insulin resistance. The mice were then inoculated with 1x109pfu of either
AdshRNAControl (group 1) or AdshRNABIM (group 2), this treatment resulted in a
>80% reduction in BIM expression in the liver of AdshRNABIM treated mice (Fig.
6H). Strikingly, after 7 days of adenovirus infection fat accumulation was reduced in
BIM knockdown livers as compared to AdshRNAControl (Figs. 4I-J). Moreover,
insulin tolerance was significantly improved in HFF mice treated with AdshRNABIM
(Fig. 6K). This result demonstrates that BIM is a master regulator of obesity driven
insulin resistance in the liver and has potential as a therapeutic target for novel anti
diabetogenic drugs.
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DISCUSSION.
NAFLD is a common cause of chronic liver dysfunction in obesity, and previous studies suggested an important role for JNK/c Jun in hepatic steatosis (4 7). Indeed, a strong correlation has been observed between the grade of steatosis and c Jun phosphorylation/activity in patients with NAFLD (46). Additionally, activated JNK was demonstrated in liver biopsies from patients with nonalcoholic steatohepatitis
(NASH) (47). We found in the present study that JNK/c Jun activation is responsible for free fatty acid mediated induction of BIM, and observed a trend to BIM up regulation in a small cohort of patients with different degrees of NAFLD. The key findings of our study are summarized in Fig. 7. Thus, the present in vivo and in vitro data demonstrate a novel metabolic role for BIM in the tight regulation of mitochondrial activity in hepatocytes under lipotoxic stress.
Compelling evidence links oxidative stress to insulin resistance in humans and rodent models (48 50). In obesity, reduced fatty acid oxidation and excess free fatty acids generate reducing equivalents that exceed the respiratory demand, resulting in high
– mitochondrial O2• and H2O2 generation (50; 51). ER stress and the unfolded protein response in obesity, together with the enhanced expression and activation of
NAD(P)H oxidases, contribute further to ROS generation. Our data postulate BIM as an important contributor of saturated fatty acids induced mitochondrial dysfunction and oxidative stress in fatty livers (Fig. 7). In agreement with this, neuroblastoma cells conditionally expressing BIM have increased accumulation of mitochondrial
ROS and reduced mitochondrial respiration (38).
PTPs are enzymes that dephosphorylate tyrosine phosphorylated proteins to counteract the actions of protein tyrosine kinases (40). The architecture and low thiol
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pKa of the cysteine residue in the active site of PTPs renders these proteins
susceptible to ROS. We recently demonstrated that PTP oxidation occurs in vivo in
the liver under obesity, leading to STAT1 activation and steatosis (25). We observed
reduced PTP oxidation/STAT1 phosphorylation in obese BLKO mice and decreased
expression of the downstream transcription factor PPAR γ, which promotes hepatic
lipogenesis via SREBP1/ACC1/SCD1 induction, and free fatty acid uptake via CD36
(42; 52 54). Importantly, liver biopsies from patients with NAFLD show higher levels
of PPAR γ/CD36 compared to samples from control subjects, highlighting the clinical
relevance of this pathway (55; 56). Thus, the decreased steatosis observed in BLKO
mice may result from the lower STAT1/PPAR γ activity, occurring as a direct
consequence of reduced oxidative stress and PTP oxidation, resulting in decreased
lipogenesis via SREBP1/SCD1/ACC1 and free fatty acid uptake via CD36 (Fig. 7). In
addition, we observed enhanced β oxidation and serum β hydroxybutyrate in obese
BLKO mice, probably due to enhanced mitochondrial activity in the hepatocytes,
which also contributed to reduced steatosis in these mice. Interestingly, our data
indicate that the improved hepatic lipid metabolism in BLKO mice is accompanied by
enhanced energy expenditure, a link that has also been shown in alternative mouse
models of obesity (25; 57). Further studies to clarify this cross talk are clearly
warranted.
One caveat of our study is that we found more than 500 genes with metabolic
associated functions differentially expressed in BIM deficient hepatocytes. Thus, we
cannot exclude additional mechanisms contributing to the observed phenotype in
these mice which, based on our gene hits, can be assessed in future studies.
Nonetheless, our striking results showing a reversal of the damaged liver phenotype in
obese mice targeted with BIM knockdown constructs strongly supports the idea that
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BIM plays a central role in the regulation of cellular metabolism and liver dysfunction in obesity.
In conclusion, we link obesity with liver steatosis and ROS through the identification of a novel JNK/c Jun/BIM axis. Our data highlight the potential utility of targeted pharmacological approaches towards BIM in combating the associated hepatic complications of obesity.
ACKNOWLEDGMENTS
We thank Evan Pappas, Christina Tan, Cameron Kos and Stacey Fynch (St Vincent’s
Institute, Australia) for excellent technical assistance, Philippe Bouillet (The Walter and Eliza Hall Institute, Australia) for the Bimlox/lox mice, Jibran Wali, Thomas Kay and Shiang Lim (St Vincent’s Institute, Australia) for reagents, protocols and helpful discussions, and Latifa Bakiri (Centro Nacional de Investigaciones Oncológicas,
Spain) and Shane Grey (Garvan Institute of Medical Research, Australia) for critical reading of the manuscript. The authors declare no conflict of interest.
FUNDING
This work was supported by a National Health and Medical Research Council of
Australia (NHMRC) project grant (APP1071350). ENG is supported by a Juvenile
Diabetes Research Foundation (JDRF) fellowship. The St Vincent’s Institute receives support from the Operational Infrastructure Support Scheme of the Government of
Victoria.
AUTHOR CONTRIBUTIONS
20 Page 21 of 62 Diabetes
SAL, LP, SG, MIE, WJS, JVT, KL, HET, AS, DJG, JBdH researched data and result
analysis. ET, CM provided human liver biopsies, contributed to discussion and
reviewed and edited the manuscript. DLE contributed to data analysis and discussion
and reviewed and edited the manuscript. ENG researched data; designed experiments;
and reviewed, edited, and wrote the manuscript. ENG is the guarantor of this work
and, as such, had full access to all the data in the study and take responsibility for the
integrity of the data and the accuracy of the data analysis.
REFERENCES
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FIGURE LEGENDS
Figure 1. Saturated free fatty acids induce BIM expression in hepatocytes through
JNK/c-Jun activation. A: Liver extracts from chow fed versus 16 week HFF
C57BL/6 male mice were processed and then analysed by immunoblotting as
indicated (n=6). B: Primary hepatocytes isolated from chow vs. 16 week HFF
C57BL/6 male mice were processed and then analysed by immunoblotting as
indicated (n=2 3). Quantification of BIM band intensities is indicated at the bottom as
a ratio to the loading control β actin. C: Box plot of median expression of BIM
related to the house keeping protein β actin in liver biopsies from NAFLD patients
and non tumorigenic healthy liver controls. D: Primary hepatocytes isolated from 8
to 12 week old C57BL/6 mice were treated with 0.5mM palmitate at different time
points, and processed for immunoblot analysis as indicated (n=6). E: Liver extracts
from chow versus 16 week HFF C57BL/6 male mice were processed and then
analysed by immunoblotting as indicated (n=6). F-G: Primary hepatocytes isolated
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from 8 to 12 week old C57BL/6 mice were treated with 0.5mM palmitate at different time points, and processed for immunoblot analysis (F) or qPCR (G) as indicated
(n=3 4). H: Primary hepatocytes were treated with 0.5mM palmitate and the JNK inhibitor SP600125 (10 M) at different time points, and processed for immunoblot analysis (n=3). I: Primary hepatocytes were transfected with scramble control or
JNK specific siRNAs prior to palmitate treatment and processed for immunoblot analysis as indicated. The result is representative of two independent experiments.
Quantification of band intensities is indicated at the bottom as a ratio to the loading control β actin. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.
Figure 2. Liver-specific BIM knockout (BLKO) mice were generated in a C57BL/6 background. A: BIMlox/lox (C57BL/6) were mated with Alb Cre (C57BL/6) mice for the conditional deletion of BIM in hepatocytes. BLKO and control male mice were maintained on a chow diet for 28 weeks and body composition determined with an
EchoMRI scan (n=10 11). B: Gonadal white adipose tissue (WAT), liver, and gastrocnemius muscle relative weights were determined after chow feeding (n=8 10).
C: Fat percentage in livers from 28 week old BLKO and control mice determined with an EchoMRI scan (n=5). D: Insulin tolerance test (0.65mU/g body weight; i.p.) were conducted after 25 weeks of chow feeding (n=12 14). E: Pyruvate tolerance tests (2mg sodium pyruvate/g body weight; i.p.) were conducted after 27 weeks of chow feeding (n=9). *P < 0.05, **P < 0.01 vs. control.
Figure 3. Hepatic deficiency of BIM reduces liver weight and improves insulin sensitivity in obese mice. A: 8 9 week old male lox/lox and BLKO mice were HFF for 14 weeks. Body weights were measured on a weekly basis, and the incremental increase in body weight was determined (n=21 23). B-C: Body composition (n=8 11)
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and tissue weights (n=9 19) in 18 week HFF lox/lox and BLKO mice. D-E: lox/lox
and BLKO male mice were fed a high fat diet for 16 18 weeks, livers extracted, fixed
in formalin, paraffin embedded, and processed for histology (haematoxylin & eosin).
Scale bars are indicated. F: Fat percentage in livers from 20 week HFF BLKO and
control mice (n=8 9). G: 19 week HFF BLKO and control mice were fasted
overnight, and triacylglycerides (TAG) quantified in serum (n=15 16). H-J: Glucose
(2mg/g, n=9 10, H), insulin (0.65mU/g, n=8 11, I) and pyruvate (2mg/g, n=10 13, J)
tolerance tests were conducted in HFF BLKO and control mice and the areas under
the curve (AUC) calculated. K: HFF BLKO and control mice were injected with PBS
or insulin (0.65mU insulin/g body weight, 10 min). Livers extracted and processed for
immunoblotting with the indicated antibodies (n=3 7). L-M: Serum insulin levels
(n=8 11, K) and pancreas insulin area per islet (n=3, L) in HFF BLKO and control
mice. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.
Figure 4. Saturated free fatty acids induce BIM translocation to the mitochondria.
A: Confocal microscopy analysis of BIM distribution in frozen livers from chow fed
versus 16 week HFF C57BL/6 male mice. The profile of fluorescence intensity and
co localization was analysed using quantification software (Nikon). Images are
representative of two independent experiments. Scale bars are indicated. B: Confocal
microscopy of BIM distribution in 0.5mM palmitate treated or control primary
hepatocytes (8h treatment). The profile of fluorescence intensity and co localization
was analysed using quantification software (Nikon). Images are representative of
three independent experiments. Scale bars are indicated.
Figure 5. BIM deficiency decreases oxidative stress and increases fatty acid
oxidation. A: Western blot showing up regulation of BIM in palmitate treated
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hepatocytes and deficiency of the protein in BLKO cells. MitoSox Red staining
(1.2 M) of BLKO and control hepatocyte cells treated with 0.5mM palmitate for 12h.
Quantification shows the corrected total cell fluorescence in the hepatocytes determined by image analysis. Results are expressed as mean ± SEM (n=4 7). Bar,
100 m. B: The cell permeant reagent 2’,7’ –dichlorofluorescin diacetate (0.2 M,
DCFDA), which is later oxidized by ROS into 2’, 7’–dichlorofluorescein (DCF) and detected by FACS, has been used to measure cellular ROS formation induced by 12h treatment with palmitate in BLKO and control hepatocytes (n=6). C: Livers were harvested from 4 h fasted mice, and processed for nitrotyrosine staining (n=5). D:
Liver extracts from HFF BLKO and control mice were processed in presence of
NEM, DTT, applied to a gel filtration column, treated with pervanadate, and then analysed by immunoblotting with an PTPox and p Tyr antibodies. E-F: Livers were harvested from 4 h fasted mice, and processed for immunoblot analysis as indicated
(n=6 10). G: Livers were harvested from 4 h fasted mice, and processed for qPCR analysis as indicated (n=7 10). H: Lipid oxidation determined by indirect calorimetry
(n=8). I: β hydroxybutyrate levels in serum from fed BLKO and lox/lox obese mice
(20 weeks on high fat diet, n=9). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.
Figure 6. Altered insulin signalling upon hepatic reduction of BIM in obesity. A:
Immunoblot analysis of BIM expression in liver samples. B-C: Glucose (2mg/g, B) and insulin tolerance (0.65mU/g, C) tests were conducted in HFF BLKO heterozygous and control mice (n=6). D: 19 week old lox/lox mice were infected with either AdControl or AdCRE (1x109pfu) and 4 weeks later the liver was extracted, processed and analysed by immunoblotting as indicated (n=4). Body weights: 33.5±1.7g, no differences between groups. E: Insulin tolerance tests
(0.65mU/g) of lox/lox mice 4 weeks after adenovirus infection as indicated in F
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(n=6). F: Expression of GFP (live microscopy fluorescence), and BIM (Western blot)
in mouse hepatocytes 48h after infection with 2x106pfu/mL of AdshRNABIM or
AdshRNAControl (n=2). Bar, 100 m. G: Total palmitate oxidation measured in
hepatocytes isolated from mice 48h after infection with 2x106pfu/mL of
AdshRNABIM or AdshRNAControl (n=3). H: Liver extracts from 20 week HFF
C57BL/6 mice were infected with either AdshRNABIM or AdshRNAControl
(1x109pfu), and 7 days later processed and analysed by immunoblotting as indicated
(n=5). I: Livers from 7 day infected mice with AdshRNABIM and AdshRNAControl
were extracted, fixed in formalin, paraffin embedded, and processed for histology
(haematoxylin & eosin). J: Fat percentage in livers from 20 week HFF C57BL/6 mice
infected with either AdshRNABIM or AdshRNAControl (1x109pfu, n=6 7). K:
Insulin tolerance tests (0.65mU/g) of obese mice before and 5 days after adenovirus
infection as indicated (n=7). Body weights: 43.1±1g, no differences between groups.
*P < 0.05, **P < 0.01, ***P < 0.001 vs. control.
Figure 7. Proposed model of the role of BIM in liver steatosis. In addition to its
known function as a potent inducer of apoptosis, BIM is involved in regulation of
mitochondrial activity and glucose homeostasis in hepatocytes. The present data
clarify a novel mechanism whereby obesity triggers activation of the BCL 2 family
member BIM, resulting in liver damage and insulin resistance. Further, we are able to
rescue obese mice from liver dysfunction by short term modulation of hepatocyte
expressed BIM.
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Supplemental Data JNK activation of BIM promotes hepatic oxidative stress, steatosis and insulin resistance in obesity. Sara A. Litwak, Lokman Pang, Sandra Galic, Mariana Igoillo-Esteve, William J. Stanley, Jean-Valery Turatsinze, Kim Loh, Helen E. Thomas, Arpeeta Sharma, Eric Trepo, Christophe Moreno, Daniel J. Gough, Decio L. Eizirik, Judy B. de Haan, Esteban N. Gurzov.
Supplementary Table 1. Differential gene expression in BLKO and lox/lox hepatocytes (pValue < 0.05). geneID geneName biotype logFC PValue ENSMUSG00000014599 'Csf1 protein_coding 16.46136649 3.23E-05 ENSMUSG00000035530 'Eif1 protein_coding 15.98627321 0.001824962 ENSMUSG00000002320 'Tm9sf1 protein_coding 15.91894903 5.31E-05 ENSMUSG00000038650 'Rnh1 protein_coding 15.50305059 0.002305602 ENSMUSG00000036427 'Gpi1 protein_coding 15.279278 0.002572604 ENSMUSG00000002588 'Pon1 protein_coding 15.21583542 0.002636011 ENSMUSG00000003072 'Atp5d protein_coding 15.21341406 0.000101955 ENSMUSG00000041355 'Ssr2 protein_coding 15.12996528 0.000107201 ENSMUSG00000004270 'Lpcat3 protein_coding 14.93288827 0.000123898 ENSMUSG00000044894 'Uqcrq protein_coding 14.93140465 0.000123327 ENSMUSG00000021492 'F12 protein_coding 14.86899797 0.00297139 ENSMUSG00000017002 'Slpi protein_coding 14.78299748 0.003143925 ENSMUSG00000029776 'Hibadh protein_coding 14.7627766 0.000139486 ENSMUSG00000020124 'Usp15 protein_coding 14.66884921 0.000147357 ENSMUSG00000015120 'Ube2i protein_coding 14.65530632 0.003234466 ENSMUSG00000028837 'Psmb2 protein_coding 14.63887475 0.000149997 ENSMUSG00000029465 'Arpc3 protein_coding 14.61395479 0.00324177 ENSMUSG00000002992 'Apoc2 protein_coding 14.55240879 0.018768027 ENSMUSG00000034902 'Pip5k1c protein_coding 14.43779877 0.000165322 ENSMUSG00000005142 'Man2b1 protein_coding 14.42900955 0.000165677 ENSMUSG00000052296 'Ppp6r1 protein_coding 14.41761101 0.00340735 ENSMUSG00000024120 'Lrpprc protein_coding 14.34230669 0.003462007 ENSMUSG00000030522 'Mtmr10 protein_coding 14.33684483 0.000173591 ENSMUSG00000069922 'Ces3a protein_coding 14.32243073 0.003543652 ENSMUSG00000055912 'Tmem150a protein_coding 14.31553368 0.000169368 ENSMUSG00000030127 'Cops7a protein_coding 14.29070084 0.003486303 ENSMUSG00000040028 'Elavl1 protein_coding 14.28421041 0.000169768 ENSMUSG00000004455 'Ppp1cc protein_coding 14.25458875 0.003488146 ENSMUSG00000027618 'Nfs1 protein_coding 14.22579709 0.003775546 ENSMUSG00000063659 'Zbtb18 protein_coding 14.20535039 0.000175615 ENSMUSG00000029561 'Oasl2 protein_coding 14.19862819 0.003936564 ENSMUSG00000039197 'Adk protein_coding 14.18660604 0.003533487 ENSMUSG00000016481 'Cr1l protein_coding 14.15445727 0.003559495 ENSMUSG00000035227 'Spcs2 protein_coding 14.1075008 0.003581289 ENSMUSG00000030647 'Ndufc2 protein_coding 14.06528397 0.000181126 ENSMUSG00000064080 'Fbln2 protein_coding 14.00940958 0.019781123 ENSMUSG00000036046 '5031439G07Rik protein_coding 13.9879808 0.003631101 ENSMUSG00000042102 'Dmgdh protein_coding 13.97914321 0.000184724 ENSMUSG00000068856 'Sf3b4 protein_coding 13.92800248 0.003619958 ENSMUSG00000036197 'Gxylt1 protein_coding 13.92747478 0.00018308 ENSMUSG00000052151 'Plpp2 protein_coding 13.91579112 0.019878983 ENSMUSG00000063480 'Nhp2l1 protein_coding 13.89779154 0.003620479 ENSMUSG00000054509 'Parp4 protein_coding 13.87994775 0.003619723 ENSMUSG00000028076 'Cd1d1 protein_coding 13.85990129 0.000188006 ENSMUSG00000042524 'Sun2 protein_coding 13.85441885 0.00365374 ENSMUSG00000042423 'Fbrs protein_coding 13.8436049 0.000186325 ENSMUSG00000025371 'Chmp6 protein_coding 13.83634997 0.00018626 ENSMUSG00000028937 'Acot7 protein_coding 13.83445489 0.00364304 ENSMUSG00000009563 'Tor2a protein_coding 13.80150595 0.003657859 ENSMUSG00000040940 'Arhgef1 protein_coding 13.79731615 0.003651211 ENSMUSG00000027998 'Plrg1 protein_coding 13.73169535 0.003696087 ENSMUSG00000025722 'Wdr73 protein_coding 13.73042802 0.000191821 ENSMUSG00000027652 'Ralgapb protein_coding 13.72711924 0.003687387 ENSMUSG00000032536 'Trak1 protein_coding 13.72258246 0.000190095 ENSMUSG00000075701 'Vimp protein_coding 13.7215916 0.00368105 ENSMUSG00000029999 'Tgfa protein_coding 13.71412686 0.003666851 ENSMUSG00000045098 'Suv420h1 protein_coding 13.69150369 0.000190934 ENSMUSG00000040263 'Klhdc4 protein_coding 13.68297977 0.000190813 ENSMUSG00000029621 'Arpc1a protein_coding 13.68231678 0.003680093 ENSMUSG00000026153 'Fam135a protein_coding 13.64338824 0.003681504 ENSMUSG00000042744 'Gm15800 protein_coding 13.63267208 0.003675412 ENSMUSG00000033285 'Wdr3 protein_coding 13.63095096 0.003683217 ENSMUSG00000027598 'Itch protein_coding 13.61882343 0.003675974 ENSMUSG00000052459 'Atp6v1a protein_coding 13.61848076 0.019899045 ENSMUSG00000038172 'Ttc39b protein_coding 13.59917749 0.003683594 ENSMUSG00000103818 'Gm38009 TEC 13.58385509 0.020027277 ENSMUSG00000028140 'Mrpl9 protein_coding 13.58261143 0.00368632 ENSMUSG00000083885 'Gm13213 processed_pseudogene 13.56384333 0.019958789 ENSMUSG00000029162 'Khk protein_coding 13.56200584 0.003678335 ENSMUSG00000018559 'Ctdnep1 protein_coding 13.55921088 0.003673612 ENSMUSG00000024083 'Pja2 protein_coding 13.54311953 0.003674528 ENSMUSG00000025138 'Sirt7 protein_coding 13.54272568 0.019893027 Diabetes Page 38 of 62
ENSMUSG00000028049 'Scamp3 protein_coding 13.522474 0.003666435 ENSMUSG00000059447 'Hadhb protein_coding 13.52209012 0.019999462 ENSMUSG00000025858 'Get4 protein_coding 13.50406268 0.003664773 ENSMUSG00000034201 'Gas2l1 protein_coding 13.48971381 0.003636429 ENSMUSG00000000743 'Chmp1a protein_coding 13.4880159 0.019828859 ENSMUSG00000004610 'Etfb protein_coding 13.47965471 0.003655471 ENSMUSG00000047797 'Gjb1 protein_coding 13.46490376 0.003766409 ENSMUSG00000017858 'Ift52 protein_coding 13.45069861 0.000188307 ENSMUSG00000006392 'Med8 protein_coding 13.44922909 0.003649085 ENSMUSG00000003308 'Keap1 protein_coding 13.42226948 0.003635892 ENSMUSG00000022965 'Ifngr2 protein_coding 13.41721617 0.019774597 ENSMUSG00000028646 'Rragc protein_coding 13.4119006 0.01983329 ENSMUSG00000031256 'Cstf2 protein_coding 13.40832863 0.003639296 ENSMUSG00000025139 'Tollip protein_coding 13.3926266 0.019796664 ENSMUSG00000031403 'Dkc1 protein_coding 13.38139067 0.003637555 ENSMUSG00000022214 'Dcaf11 protein_coding 13.36930438 0.019790941 ENSMUSG00000037669 'Ldah protein_coding 13.36274254 0.003613218 ENSMUSG00000066735 'Vkorc1l1 protein_coding 13.35224325 0.003758044 ENSMUSG00000010025 'Aldh3a2 protein_coding 13.34944883 0.019783967 ENSMUSG00000019984 'Med23 protein_coding 13.3468375 0.000187288 ENSMUSG00000004040 'Stat3 protein_coding 13.34037868 0.019700191 ENSMUSG00000041258 'Zfp236 protein_coding 13.3277904 0.003629867 ENSMUSG00000024158 'Hagh protein_coding 13.32546917 0.00361054 ENSMUSG00000047824 'Pygo2 protein_coding 13.32507643 0.003614126 ENSMUSG00000028064 'Sema4a protein_coding 13.30465859 0.003623161 ENSMUSG00000040356 'Skiv2l protein_coding 13.28103333 0.003629245 ENSMUSG00000026966 'Ssna1 protein_coding 13.27897556 0.000188605 ENSMUSG00000029066 'Mrpl20 protein_coding 13.26566685 0.003625469 ENSMUSG00000031357 'Syap1 protein_coding 13.25615523 0.00364217 ENSMUSG00000031886 'Ces2e protein_coding 13.24997787 0.019790426 ENSMUSG00000042997 'Nhlrc3 protein_coding 13.24917026 0.003632358 ENSMUSG00000024218 'Taf11 protein_coding 13.23719274 0.000186702 ENSMUSG00000026439 'Rbbp5 protein_coding 13.17960572 0.003639331 ENSMUSG00000024773 'Atg2a protein_coding 13.1605898 0.019698705 ENSMUSG00000053898 'Ech1 protein_coding 13.15881698 0.003644639 ENSMUSG00000033124 'Atg9a protein_coding 13.15625135 0.019744752 ENSMUSG00000027498 'Cstf1 protein_coding 13.13720192 0.003652294 ENSMUSG00000027429 'Sec23b protein_coding 13.12462327 0.01970545 ENSMUSG00000005687 'Bcas2 protein_coding 13.08637714 0.003655898 ENSMUSG00000058254 'Tspan7 protein_coding 13.07709422 0.000209523 ENSMUSG00000074796 'Slc4a11 protein_coding 13.07031007 0.00366264 ENSMUSG00000030533 'Unc45a protein_coding 13.06230139 0.003689699 ENSMUSG00000013160 'Atp6v0d1 protein_coding 13.04331974 0.019940528 ENSMUSG00000024889 'Rce1 protein_coding 13.03897653 0.000195071 ENSMUSG00000074207 'Adh1 protein_coding 13.03254443 0.020006355 ENSMUSG00000033016 'Nfatc1 protein_coding 13.02741271 0.000195053 ENSMUSG00000023055 'Calcoco1 protein_coding 13.02030493 0.003707009 ENSMUSG00000092137 'Gcom1 protein_coding 13.01673503 0.020024285 ENSMUSG00000024805 'Pcgf5 protein_coding 13.01301535 0.000216078 ENSMUSG00000032387 'Rbpms2 protein_coding 13.0061069 0.020026967 ENSMUSG00000037949 'Ano10 protein_coding 13.00071758 0.003723809 ENSMUSG00000022828 'Gtf2e1 protein_coding 12.9972606 0.003699992 ENSMUSG00000030619 'Eed protein_coding 12.98452204 0.000206034 ENSMUSG00000003199 'Mpnd protein_coding 12.97419459 0.003727538 ENSMUSG00000001755 'Coasy protein_coding 12.97405652 0.003732301 ENSMUSG00000051234 'Rnf7 protein_coding 12.96612754 0.003719797 ENSMUSG00000024785 'Rcl1 protein_coding 12.95815649 0.020092175 ENSMUSG00000021796 'Bmpr1a protein_coding 12.94772393 0.020226747 ENSMUSG00000035914 'Cd276 protein_coding 12.93908998 0.020136528 ENSMUSG00000034729 'Mrps10 protein_coding 12.92287256 0.003756197 ENSMUSG00000031878 'Nae1 protein_coding 12.92106693 0.020196944 ENSMUSG00000029472 'Anapc5 protein_coding 12.91194207 0.02014867 ENSMUSG00000039456 'Morc3 protein_coding 12.91189423 0.003745009 ENSMUSG00000027968 'Larp7 protein_coding 12.91165438 0.000200441 ENSMUSG00000025451 'Paip1 protein_coding 12.88610555 0.02020652 ENSMUSG00000028563 'Tm2d1 protein_coding 12.87638224 0.000203677 ENSMUSG00000046722 'Cdc42se1 protein_coding 12.86339684 0.020252386 ENSMUSG00000022820 'Ndufb4 protein_coding 12.8581685 0.003784932 ENSMUSG00000066072 'Cyp4a10 protein_coding 12.84572827 0.003883008 ENSMUSG00000041135 'Ripk2 protein_coding 12.83978399 0.020366356 ENSMUSG00000040268 'Plekha1 protein_coding 12.83920877 0.003824364 ENSMUSG00000021751 'Acox2 protein_coding 12.83596137 0.020455311 ENSMUSG00000073889 'Il11ra1 protein_coding 12.82853938 0.000312427 ENSMUSG00000026223 'Itm2c protein_coding 12.82810778 0.02054789 ENSMUSG00000001829 'Clpb protein_coding 12.8279686 0.003852943 ENSMUSG00000032577 'Mapkapk3 protein_coding 12.81814039 0.020577627 ENSMUSG00000034255 'Arhgap27 protein_coding 12.8052791 0.003850316 ENSMUSG00000041733 'Coq5 protein_coding 12.80165205 0.003853134 ENSMUSG00000026319 '2310035C23Rik protein_coding 12.77995892 0.003866185 ENSMUSG00000028672 'Hmgcl protein_coding 12.77900359 0.020544187 ENSMUSG00000040785 'Ttc3 protein_coding 12.76539512 0.020610327 ENSMUSG00000001285 'Myg1 protein_coding 12.7637857 0.020694459 ENSMUSG00000049152 'Ugt3a2 protein_coding 12.7505063 0.020740008 Page 39 of 62 Diabetes
ENSMUSG00000110104 'RP24-243J4.5 protein_coding 12.7372061 0.020802215 ENSMUSG00000024639 'Gnaq protein_coding 12.72640171 0.003940299 ENSMUSG00000025245 'Lztfl1 protein_coding 12.72542338 0.003944847 ENSMUSG00000004561 'Mettl17 protein_coding 12.72047588 0.000220423 ENSMUSG00000034157 'Cipc protein_coding 12.71777018 0.020871516 ENSMUSG00000051043 'Gprc5c protein_coding 12.7108239 0.02084638 ENSMUSG00000015363 'Trabd protein_coding 12.70707188 0.020902225 ENSMUSG00000001964 'Emd protein_coding 12.7052811 0.020909343 ENSMUSG00000093989 'Rnasek protein_coding 12.69894182 0.020874721 ENSMUSG00000093392 'Gm6061 transcribed_processed_pseudogene 12.69569251 0.020932805 ENSMUSG00000078789 'Dph1 protein_coding 12.67487997 0.021071372 ENSMUSG00000033475 'Tomm6 protein_coding 12.66346727 0.021185895 ENSMUSG00000028132 'Tmem56 protein_coding 12.6553373 0.021154556 ENSMUSG00000039956 'Mrap protein_coding 12.6434659 0.004026202 ENSMUSG00000018931 'Natd1 protein_coding 12.63334382 0.00409415 ENSMUSG00000107283 'Mpv17 protein_coding 12.63217604 0.021335188 ENSMUSG00000073555 'Gm4951 protein_coding 12.62329928 0.021377633 ENSMUSG00000021786 'Oxsm protein_coding 12.6202696 0.004060597 ENSMUSG00000046761 'Fam83h protein_coding 12.61180546 0.021411387 ENSMUSG00000026922 'Agpat2 protein_coding 12.59250226 0.021424328 ENSMUSG00000030674 'Qprt protein_coding 12.59149062 0.004109118 ENSMUSG00000029802 'Abcg2 protein_coding 12.58864369 0.021448113 ENSMUSG00000041084 'Ostc protein_coding 12.58643325 0.021504028 ENSMUSG00000038268 'Ovca2 protein_coding 12.58643325 0.021503242 ENSMUSG00000041845 'Rhod protein_coding 12.5741343 0.021632526 ENSMUSG00000007721 'Ccdc124 protein_coding 12.56788855 0.021609092 ENSMUSG00000034432 'Cops8 protein_coding 12.56265278 0.021665779 ENSMUSG00000028991 'Mtor protein_coding 12.56134086 0.02167139 ENSMUSG00000026095 'Asnsd1 protein_coding 12.54219666 0.021891391 ENSMUSG00000030879 'Mrpl17 protein_coding 12.53217264 0.02186799 ENSMUSG00000040128 'Pnrc1 protein_coding 12.5200681 0.021935773 ENSMUSG00000033623 'Pcgf3 protein_coding 12.51465554 0.021958548 ENSMUSG00000033216 'Eefsec protein_coding 12.51306355 0.004231497 ENSMUSG00000020280 'Pus10 protein_coding 12.51209721 0.004232604 ENSMUSG00000027575 'Arfgap1 protein_coding 12.50125394 0.02214235 ENSMUSG00000032540 'Abhd5 protein_coding 12.49570605 0.022159871 ENSMUSG00000022678 'Nde1 protein_coding 12.48435631 0.004290252 ENSMUSG00000030059 'Tmf1 protein_coding 12.48314476 0.022231131 ENSMUSG00000006356 'Crip2 protein_coding 12.48314476 0.022231973 ENSMUSG00000041774 'Ydjc protein_coding 12.48188572 0.00428761 ENSMUSG00000022848 'Dirc2 protein_coding 12.47201916 0.004298078 ENSMUSG00000019590 'Cyb561 protein_coding 12.4720051 0.022196481 ENSMUSG00000031556 'Tm2d2 protein_coding 12.46747168 0.004298037 ENSMUSG00000040584 'Abcb1a protein_coding 12.46409534 0.022332991 ENSMUSG00000020840 'Blmh protein_coding 12.4548329 0.022408101 ENSMUSG00000042272 'Sestd1 protein_coding 12.45193356 0.022383785 ENSMUSG00000024792 'Zfpl1 protein_coding 12.45122143 0.022393339 ENSMUSG00000021252 '0610007P14Rik protein_coding 12.44836935 0.022425313 ENSMUSG00000020258 'Glyctk protein_coding 12.44765545 0.022432355 ENSMUSG00000001666 'Ddt protein_coding 12.43666684 0.022490564 ENSMUSG00000034591 'Slc41a2 protein_coding 12.43487059 0.004384544 ENSMUSG00000060012 'Kif13b protein_coding 12.42764968 0.004384604 ENSMUSG00000040771 'Oard1 protein_coding 12.42731023 0.004395672 ENSMUSG00000028560 'Usp1 protein_coding 12.41873037 0.004408366 ENSMUSG00000000378 'Ccm2 protein_coding 12.40190782 0.022756066 ENSMUSG00000028024 'Enpep protein_coding 12.40030798 0.000268697 ENSMUSG00000015247 'Nipsnap3b protein_coding 12.39160999 0.022828225 ENSMUSG00000030423 'Pop4 protein_coding 12.39057927 0.004470814 ENSMUSG00000033416 'Gucd1 protein_coding 12.39051919 0.022933375 ENSMUSG00000010609 'Psen2 protein_coding 12.38633903 0.004473392 ENSMUSG00000030630 'Fah protein_coding 12.38302605 0.022987079 ENSMUSG00000026209 'Dnpep protein_coding 12.36915148 0.023050448 ENSMUSG00000036305 'Rpl39-ps transcribed_processed_pseudogene 12.368584 0.004523615 ENSMUSG00000038206 'Fbxo8 protein_coding 12.36142037 0.004515328 ENSMUSG00000018425 'Dhx40 protein_coding 12.35572382 0.023204775 ENSMUSG00000020166 'Cnot2 protein_coding 12.3526581 0.023252537 ENSMUSG00000018401 'Mtmr4 protein_coding 12.35112279 0.023273344 ENSMUSG00000025962 'Fastkd2 protein_coding 12.34864818 0.023270367 ENSMUSG00000033653 'Vps8 protein_coding 12.34200403 0.004606579 ENSMUSG00000020829 'Slc46a1 protein_coding 12.33257052 0.023572014 ENSMUSG00000007833 'Aldh16a1 protein_coding 12.32992547 0.004656517 ENSMUSG00000041650 'Pcca protein_coding 12.32837032 0.000288617 ENSMUSG00000026405 'C4bp protein_coding 12.30803432 0.023901026 ENSMUSG00000099908 'Gm28539 protein_coding 12.30674617 0.023968898 ENSMUSG00000037415 'Ranbp10 protein_coding 12.30333336 0.023997471 ENSMUSG00000029433 'Diablo protein_coding 12.29568106 0.024171173 ENSMUSG00000050714 'Zbtb26 protein_coding 12.28541334 0.005044514 ENSMUSG00000004565 'Pnpla6 protein_coding 12.27893081 0.004923433 ENSMUSG00000062980 'Cped1 protein_coding 12.26677461 0.024662849 ENSMUSG00000021838 'Samd4 protein_coding 12.26183411 0.005004155 ENSMUSG00000025495 'Ptdss2 protein_coding 12.25962771 0.005023386 ENSMUSG00000028715 'Cyp4a14 protein_coding 12.2570832 0.024807096 ENSMUSG00000031149 'Praf2 protein_coding 12.24234244 0.025028286 Diabetes Page 40 of 62
ENSMUSG00000026675 'Hsd17b7 protein_coding 12.24078476 0.025009104 ENSMUSG00000000751 'Rpa1 protein_coding 12.23077015 0.025271387 ENSMUSG00000006315 'Tmem147 protein_coding 12.22100116 0.025515058 ENSMUSG00000048410 'Zfp407 protein_coding 12.2189915 0.005291231 ENSMUSG00000075415 'Fnbp1 protein_coding 12.21726656 0.000347153 ENSMUSG00000037652 'Phc3 protein_coding 12.21264771 0.025570462 ENSMUSG00000042903 'Foxo4 protein_coding 12.21169565 0.005245874 ENSMUSG00000018906 'P4ha2 protein_coding 12.21097537 0.025610822 ENSMUSG00000053317 'Sec61b protein_coding 12.20057945 0.025811149 ENSMUSG00000015357 'Clpx protein_coding 12.19752644 0.025830782 ENSMUSG00000027509 'Rae1 protein_coding 12.19414453 0.025945935 ENSMUSG00000033365 'Ipo13 protein_coding 12.19245059 0.025996786 ENSMUSG00000024581 'Napg protein_coding 12.18866586 0.026086144 ENSMUSG00000068580 'Zfyve19 protein_coding 12.18492306 0.005398752 ENSMUSG00000038416 'Cdc16 protein_coding 12.17492876 0.026282793 ENSMUSG00000041716 'Gm20604 protein_coding 12.17369958 0.005478178 ENSMUSG00000028826 'Tmem57 protein_coding 12.16507361 0.026440391 ENSMUSG00000039656 'Rxrb protein_coding 12.16453941 0.026509588 ENSMUSG00000050552 'Lamtor4 protein_coding 12.16419716 0.005515346 ENSMUSG00000039686 'Zer1 protein_coding 12.16015875 0.005545729 ENSMUSG00000034187 'Nsf protein_coding 12.15988208 0.02659019 ENSMUSG00000008226 'Scrn3 protein_coding 12.15470014 0.00558408 ENSMUSG00000026170 'Cyp27a1 protein_coding 12.14860812 0.026987085 ENSMUSG00000032898 'Fbxo21 protein_coding 12.14464299 0.005762434 ENSMUSG00000037692 'Ahdc1 protein_coding 12.13895238 0.005691427 ENSMUSG00000029454 'Mapkapk5 protein_coding 12.13261116 0.02723585 ENSMUSG00000041225 'Arhgap12 protein_coding 12.12833436 0.027218709 ENSMUSG00000027076 'Timm10 protein_coding 12.12579193 0.027324084 ENSMUSG00000024039 'Cbs protein_coding 12.12364671 0.02740429 ENSMUSG00000040374 'Pex2 protein_coding 12.12184712 0.027422033 ENSMUSG00000028910 'Mecr protein_coding 12.12150259 0.005752847 ENSMUSG00000039236 'Isg20 protein_coding 12.12004528 0.027504221 ENSMUSG00000040824 'Snrpd2 protein_coding 12.11824118 0.027572049 ENSMUSG00000037606 'Osbpl5 protein_coding 12.1167991 0.005806381 ENSMUSG00000021501 'Caml protein_coding 12.11587705 0.027499589 ENSMUSG00000021820 'Camk2g protein_coding 12.10715839 0.005837769 ENSMUSG00000030207 'Fam234b protein_coding 12.10193738 0.005859569 ENSMUSG00000060512 '0610040J01Rik protein_coding 12.10028472 0.005871114 ENSMUSG00000025268 'Maged2 protein_coding 12.09274408 0.028046753 ENSMUSG00000006471 'Ndor1 protein_coding 12.0909055 0.028077997 ENSMUSG00000024829 'Mrpl21 protein_coding 12.08699269 0.02805881 ENSMUSG00000037788 'Vopp1 protein_coding 12.08385188 0.000434342 ENSMUSG00000038844 'Kif16b protein_coding 12.07784643 0.02826892 ENSMUSG00000058833 '2810428I15Rik protein_coding 12.0702125 0.006115686 ENSMUSG00000097108 'Gm26581 lincRNA 12.06843566 0.02855163 ENSMUSG00000024151 'Msh2 protein_coding 12.04443131 0.028948531 ENSMUSG00000051674 'Dcun1d4 protein_coding 12.03878609 0.029069545 ENSMUSG00000072969 'Armcx5 protein_coding 12.03795278 0.006291722 ENSMUSG00000034738 'Nostrin protein_coding 12.03462944 0.029279456 ENSMUSG00000032468 'Armc8 protein_coding 12.03462944 0.0292896 ENSMUSG00000064120 'Mocs1 protein_coding 12.02552738 0.029326632 ENSMUSG00000021027 'Ralgapa1 protein_coding 12.01924421 0.029523069 ENSMUSG00000018800 'Abca5 protein_coding 12.01598171 0.029481229 ENSMUSG00000032207 'Lipc protein_coding 12.01214569 0.029533309 ENSMUSG00000092203 '1110038B12Rik processed_transcript 12.01148962 0.02965618 ENSMUSG00000053175 'Bcl3 protein_coding 12.00829944 0.029593122 ENSMUSG00000028128 'F3 protein_coding 12.00759665 0.029740496 ENSMUSG00000005262 'Ufd1l protein_coding 12.00093937 0.029796216 ENSMUSG00000079563 'Pglyrp2 protein_coding 11.99977903 0.029919374 ENSMUSG00000038764 'Ptpn3 protein_coding 11.99899157 0.029874415 ENSMUSG00000041229 'Phf8 protein_coding 11.9948899 0.006515123 ENSMUSG00000075254 'Heg1 protein_coding 11.98994707 0.030132382 ENSMUSG00000020485 'Supt4a protein_coding 11.98695979 0.030037914 ENSMUSG00000046096 'BC030336 protein_coding 11.98401556 0.03021153 ENSMUSG00000073422 'H2-Ke6 protein_coding 11.98296423 0.007005542 ENSMUSG00000035781 'R3hdm4 protein_coding 11.97715461 0.03017688 ENSMUSG00000059288 'Cdyl protein_coding 11.97670695 0.006630086 ENSMUSG00000032253 'Phip protein_coding 11.96728234 0.030356244 ENSMUSG00000042842 'Serpinb6b protein_coding 11.94688901 0.030758729 ENSMUSG00000010554 'Mettl16 protein_coding 11.92910706 0.030994185 ENSMUSG00000030541 'Idh2 protein_coding 11.92506489 0.030994782 ENSMUSG00000032475 'Nck1 protein_coding 11.92097899 0.031081639 ENSMUSG00000036513 'Commd2 protein_coding 11.91918067 0.031246813 ENSMUSG00000061207 'Stk19 protein_coding 11.91506448 0.006950571 ENSMUSG00000028614 'Ndc1 protein_coding 11.91264671 0.031324496 ENSMUSG00000026189 'Pecr protein_coding 11.91006911 0.006966024 ENSMUSG00000046516 'Cox17 protein_coding 11.90773773 0.031485134 ENSMUSG00000029173 'Sepsecs protein_coding 11.89198598 0.031772818 ENSMUSG00000033294 'Noc4l protein_coding 11.8898727 0.031793492 ENSMUSG00000031767 'Nudt7 protein_coding 11.88563681 0.031874583 ENSMUSG00000031939 'Taf1d protein_coding 11.87370347 0.007179614 ENSMUSG00000022792 'Yars2 protein_coding 11.86852292 0.032088757 ENSMUSG00000034105 'Tldc1 protein_coding 11.8621297 0.007223264 Page 41 of 62 Diabetes
ENSMUSG00000018287 'Spag7 protein_coding 11.85988231 0.007229877 ENSMUSG00000071266 'Zfp946 protein_coding 11.85811531 0.007230239 ENSMUSG00000087231 'E230016M11Rik processed_transcript 11.84982151 0.007319391 ENSMUSG00000003762 'Coq8b protein_coding 11.8448604 0.032510814 ENSMUSG00000021097 'Clmn protein_coding 11.84051611 0.032592291 ENSMUSG00000024067 'Dpy30 protein_coding 11.84051611 0.032580698 ENSMUSG00000037536 'Fbxo34 protein_coding 11.84051218 0.0073393 ENSMUSG00000028708 'Mknk1 protein_coding 11.83820231 0.032704635 ENSMUSG00000029388 'Eif2b1 protein_coding 11.82803094 0.032786721 ENSMUSG00000023232 'Serinc2 protein_coding 11.82803094 0.032775094 ENSMUSG00000000058 'Cav2 protein_coding 11.82366035 0.03284113 ENSMUSG00000078566 'Bnip3 protein_coding 11.82080329 0.032979703 ENSMUSG00000005078 'Jkamp protein_coding 11.81707953 0.033000077 ENSMUSG00000083022 'Rps15a-ps6 processed_pseudogene 11.79745931 0.033447312 ENSMUSG00000028899 'Taf12 protein_coding 11.79409341 0.033504735 ENSMUSG00000079508 'Apoo protein_coding 11.78184938 0.007644824 ENSMUSG00000050188 'Lsm10 protein_coding 11.77582693 0.007696004 ENSMUSG00000020620 'Abca8b protein_coding 11.74516352 0.00787247 ENSMUSG00000043889 'Gm8399 pseudogene 11.74072848 0.034395815 ENSMUSG00000104293 'Gm38043 TEC 11.73312498 0.007979852 ENSMUSG00000017188 'Coa3 protein_coding 11.72399697 0.034494429 ENSMUSG00000038949 'Cnst protein_coding 11.71905847 0.008049027 ENSMUSG00000041361 'Myzap protein_coding 11.71679974 0.034702053 ENSMUSG00000107742 '9530085L11Rik TEC 11.71476136 0.011110535 ENSMUSG00000013646 'Sh3bp5l protein_coding 11.7123084 0.034873429 ENSMUSG00000038690 'Atp5j2 protein_coding 11.7123084 0.034885728 ENSMUSG00000037997 'Parp11 protein_coding 11.70029946 0.035145849 ENSMUSG00000104235 'Gm37589 TEC 11.68690182 0.008207866 ENSMUSG00000020650 'Bcap29 protein_coding 11.6868638 0.035417435 ENSMUSG00000038217 'Tlcd2 protein_coding 11.6868638 0.035413202 ENSMUSG00000046623 'Gjb4 protein_coding 11.68575551 0.035537885 ENSMUSG00000035245 'Eogt protein_coding 11.6833172 0.035579478 ENSMUSG00000031197 'Vbp1 protein_coding 11.67227088 0.035675354 ENSMUSG00000022973 'Synj1 protein_coding 11.67227088 0.035662768 ENSMUSG00000034528 'Hsd17b13 protein_coding 11.66968773 0.008358384 ENSMUSG00000026860 'Sh3glb2 protein_coding 11.66491869 0.035906781 ENSMUSG00000031819 'Emc8 protein_coding 11.65622024 0.03615208 ENSMUSG00000040520 'Manea protein_coding 11.65373145 0.036216372 ENSMUSG00000035139 'Secisbp2 protein_coding 11.64693001 0.036166416 ENSMUSG00000054263 'Lifr protein_coding 11.63948937 0.036325159 ENSMUSG00000061544 'Zfp229 protein_coding 11.63200707 0.000762855 ENSMUSG00000030729 'Pgm2l1 protein_coding 11.63080923 0.008670055 ENSMUSG00000065954 'Tacc1 protein_coding 11.61842999 0.03680803 ENSMUSG00000029432 'Gbas protein_coding 11.61693439 0.036719321 ENSMUSG00000026694 'Mettl13 protein_coding 11.60471205 0.037073472 ENSMUSG00000044350 'Lacc1 protein_coding 11.60424978 0.037009513 ENSMUSG00000002031 'Ift46 protein_coding 11.59914455 0.03709815 ENSMUSG00000066724 'Gm10175 transcribed_processed_pseudogene 11.59658514 0.037155747 ENSMUSG00000047423 'AI837181 protein_coding 11.58485812 0.037514526 ENSMUSG00000025075 'Habp2 protein_coding 11.57594475 0.037476699 ENSMUSG00000030652 'Coq7 protein_coding 11.57173494 0.037744034 ENSMUSG00000033021 'Gmppa protein_coding 11.57173494 0.037748808 ENSMUSG00000037638 'Zbtb42 protein_coding 11.56909592 0.037830287 ENSMUSG00000009772 'Nuak2 protein_coding 11.56645207 0.037943212 ENSMUSG00000027761 'Aadac protein_coding 11.56551275 0.037812935 ENSMUSG00000039929 'Urb1 protein_coding 11.56380336 0.038030488 ENSMUSG00000036565 'Ttyh3 protein_coding 11.55867041 0.009139445 ENSMUSG00000021460 'Auh protein_coding 11.55189995 0.009095093 ENSMUSG00000062991 'Nrg1 protein_coding 11.54972194 0.038124309 ENSMUSG00000040410 'Fbxl4 protein_coding 11.54589103 0.038247048 ENSMUSG00000031536 'Polb protein_coding 11.53974356 0.038477678 ENSMUSG00000038936 'Sccpdh protein_coding 11.53652165 0.038446861 ENSMUSG00000107116 'Gm43274 TEC 11.52839502 0.038561198 ENSMUSG00000033715 'Akr1c14 protein_coding 11.51219035 0.038821122 ENSMUSG00000052139 'Bre protein_coding 11.50947178 0.038880422 ENSMUSG00000083012 'Fam220a protein_coding 11.50392808 0.039102144 ENSMUSG00000039157 'Fam102a protein_coding 11.50150097 0.039241567 ENSMUSG00000029416 'Slc15a4 protein_coding 11.50128522 0.03908418 ENSMUSG00000024012 'Mtch1 protein_coding 11.48735094 0.039540871 ENSMUSG00000092918 'Gm24081 miRNA 11.48200062 0.039640974 ENSMUSG00000060681 'Slc9a6 protein_coding 11.47922452 0.039703768 ENSMUSG00000025525 'Apool protein_coding 11.47338965 0.039904332 ENSMUSG00000030060 'Hmces protein_coding 11.47338965 0.039890986 ENSMUSG00000034613 'Ppm1h protein_coding 11.46734345 0.009682404 ENSMUSG00000028986 'Klhl7 protein_coding 11.46365801 0.009709001 ENSMUSG00000009828 'Ick protein_coding 11.45936964 0.040238474 ENSMUSG00000032320 'Rcn2 protein_coding 11.45645578 0.04023741 ENSMUSG00000067547 'Gm7666 transcribed_processed_pseudogene 11.45508803 0.040369753 ENSMUSG00000024055 'Cyp4f13 protein_coding 11.45361405 0.040308324 ENSMUSG00000029022 'Miip protein_coding 11.44086972 0.009847514 ENSMUSG00000001604 'Tcea3 protein_coding 11.43978747 0.040561981 ENSMUSG00000037025 'Foxa2 protein_coding 11.43406456 0.040692356 ENSMUSG00000048490 'Nrip1 protein_coding 11.4335636 0.04076362 Diabetes Page 42 of 62
ENSMUSG00000055945 'Prr18 protein_coding 11.4262146 0.040941262 ENSMUSG00000098950 'Gm28036 protein_coding 11.4262146 0.040954962 ENSMUSG00000022974 'Paxbp1 protein_coding 11.42543741 0.040814954 ENSMUSG00000102919 'Gm37726 TEC 11.41615288 0.041107658 ENSMUSG00000014470 'Rnf166 protein_coding 11.41556876 0.010043416 ENSMUSG00000031750 'Il34 protein_coding 11.40802677 0.041170052 ENSMUSG00000063808 'Gpatch1 protein_coding 11.40736809 0.041273015 ENSMUSG00000040612 'Ildr2 protein_coding 11.39823903 0.04156232 ENSMUSG00000044165 'Bcl2l15 protein_coding 11.37469164 0.041872424 ENSMUSG00000073468 'Sft2d1 protein_coding 11.36867013 0.041962129 ENSMUSG00000028018 'Gstcd protein_coding 11.36564992 0.042040855 ENSMUSG00000041143 'Tmco4 protein_coding 11.36210792 0.042170186 ENSMUSG00000030689 'Ino80e protein_coding 11.34842535 0.042275889 ENSMUSG00000044252 'Osbpl1a protein_coding 11.34678209 0.042518784 ENSMUSG00000026999 'Nup35 protein_coding 11.3381797 0.042572269 ENSMUSG00000027329 'Spef1 protein_coding 11.33474159 0.019628873 ENSMUSG00000021767 'Kat6b protein_coding 11.32462088 0.010621298 ENSMUSG00000020721 'Helz protein_coding 11.3176751 0.042917957 ENSMUSG00000028669 'Pithd1 protein_coding 11.30205065 0.043153122 ENSMUSG00000090523 'Gypc protein_coding 11.29363158 0.010738559 ENSMUSG00000018861 'Fdxr protein_coding 11.27689448 0.010904579 ENSMUSG00000110234 'Timm10b protein_coding 11.27197218 0.043784352 ENSMUSG00000062627 'Mysm1 protein_coding 11.26061698 0.043862683 ENSMUSG00000022634 'Yaf2 protein_coding 11.25248395 0.044064038 ENSMUSG00000058979 'Cecr5 protein_coding 11.24921016 0.044162098 ENSMUSG00000070305 'Mpzl3 protein_coding 11.24794584 0.044276868 ENSMUSG00000001157 'Gmcl1 protein_coding 11.247625 0.04410221 ENSMUSG00000031295 'Phka2 protein_coding 11.24592893 0.044258569 ENSMUSG00000048058 'Ldlrad3 protein_coding 11.24592893 0.044243995 ENSMUSG00000026158 'Ogfrl1 protein_coding 11.23886009 0.011138837 ENSMUSG00000020230 'Prmt2 protein_coding 11.22274892 0.044839023 ENSMUSG00000032413 'Rasa2 protein_coding 11.2213012 0.044975375 ENSMUSG00000028969 'Cdk5 protein_coding 11.22128468 0.044774456 ENSMUSG00000036989 'Trim3 protein_coding 11.21793575 0.045038332 ENSMUSG00000054381 'Zfp747 protein_coding 11.21547939 0.011314832 ENSMUSG00000062031 'Athl1 protein_coding 11.20257955 0.045252941 ENSMUSG00000029715 'Pop7 protein_coding 11.19586686 0.045433405 ENSMUSG00000074800 'Gm4149 pseudogene 11.17598776 0.011525392 ENSMUSG00000035696 'Rnf38 protein_coding 11.17056167 0.04571712 ENSMUSG00000031683 'Lsm6 protein_coding 11.16648857 0.045953892 ENSMUSG00000016181 'Diexf protein_coding 11.15788717 0.046046111 ENSMUSG00000020385 'Clk4 protein_coding 11.15264012 0.046153335 ENSMUSG00000063047 'Zfp780b protein_coding 11.14716732 0.011713013 ENSMUSG00000057842 'Zfp595 protein_coding 11.1436121 0.046410807 ENSMUSG00000107023 'Gm42715 protein_coding 11.1410067 0.046259828 ENSMUSG00000106636 'Gm43813 processed_transcript 11.13678349 0.046443308 ENSMUSG00000004934 'Pias4 protein_coding 11.13471596 0.04660251 ENSMUSG00000022617 'Chkb protein_coding 11.13114209 0.046619105 ENSMUSG00000041961 'Znrf3 protein_coding 11.12963256 0.011822462 ENSMUSG00000006906 'Stambp protein_coding 11.12576462 0.046740433 ENSMUSG00000035268 'Pkig protein_coding 11.12236764 0.011878762 ENSMUSG00000002250 'Ppard protein_coding 11.1167574 0.046868473 ENSMUSG00000036352 'Ubac1 protein_coding 11.11536652 0.046839197 ENSMUSG00000048920 'Fkrp protein_coding 11.10453762 0.047084543 ENSMUSG00000058318 'Phf21a protein_coding 11.09123383 0.047466271 ENSMUSG00000022442 'Ttll1 protein_coding 11.08914847 0.047310919 ENSMUSG00000028464 'Tpm2 protein_coding 11.08184705 0.047450861 ENSMUSG00000026639 'Lamb3 protein_coding 11.07644359 0.047731925 ENSMUSG00000020935 'Dcakd protein_coding 11.07525675 0.047677709 ENSMUSG00000033720 'Sfxn5 protein_coding 11.07242912 0.012497871 ENSMUSG00000031508 'Ankrd10 protein_coding 11.0715545 0.04772778 ENSMUSG00000018239 'Zcchc10 protein_coding 11.06784273 0.047793379 ENSMUSG00000030527 'Crtc3 protein_coding 11.06525055 0.04796939 ENSMUSG00000051232 'Tmem199 protein_coding 11.06150016 0.048035303 ENSMUSG00000018509 'Cenpv protein_coding 11.05971844 0.047895662 ENSMUSG00000039783 'Kmo protein_coding 11.05226617 0.047982005 ENSMUSG00000045948 'Mrps12 protein_coding 11.02254019 0.048520098 ENSMUSG00000011837 'Snapc2 protein_coding 11.02206604 0.048403324 ENSMUSG00000097723 'Gm26848 lincRNA 11.01484951 0.048639326 ENSMUSG00000070811 'Sult2a2 protein_coding 11.01297588 0.012474446 ENSMUSG00000005897 'Nr2c1 protein_coding 11.01098874 0.048687049 ENSMUSG00000027905 'Ddx20 protein_coding 11.00794447 0.04886952 ENSMUSG00000004768 'Rab23 protein_coding 11.0040419 0.048970229 ENSMUSG00000030279 'C2cd5 protein_coding 10.99441208 0.012697928 ENSMUSG00000026304 'Rab17 protein_coding 10.99119515 0.012756869 ENSMUSG00000020003 'Pex7 protein_coding 10.98832519 0.049322114 ENSMUSG00000030876 'Mettl9 protein_coding 10.97642426 0.049517826 ENSMUSG00000091474 '2610021A01Rik protein_coding 10.97554636 0.049359904 ENSMUSG00000061759 'Armt1 protein_coding 10.97554636 0.04935404 ENSMUSG00000099703 'Gm28285 protein_coding 10.95983526 0.049712088 ENSMUSG00000030083 'Abtb1 protein_coding 10.95571154 0.049710617 ENSMUSG00000037325 'Bbs7 protein_coding 10.93156834 0.012979032 ENSMUSG00000071302 '2610044O15Rik8 protein_coding 10.86487399 0.013379437 Page 43 of 62 Diabetes
ENSMUSG00000050270 'Tmem220 protein_coding 10.83759616 0.001542202 ENSMUSG00000038055 'Dexi protein_coding 10.74030064 0.014204731 ENSMUSG00000037190 'Cyb561d2 protein_coding 10.73446409 0.014325662 ENSMUSG00000027281 'Slx4ip protein_coding 10.73103596 0.014347841 ENSMUSG00000039983 'Ccdc32 protein_coding 10.67359363 0.014679848 ENSMUSG00000004661 'Arid3b protein_coding 10.66450915 0.014834639 ENSMUSG00000027424 'Mgme1 protein_coding 10.61869568 0.015108809 ENSMUSG00000021694 'Ercc8 protein_coding 10.60888482 0.015226621 ENSMUSG00000026939 'Tmem141 protein_coding 10.51707269 0.015949792 ENSMUSG00000006575 'Rundc3a protein_coding 10.51390004 0.015933533 ENSMUSG00000090467 'Gm4613 processed_pseudogene 10.44681289 0.016387961 ENSMUSG00000030278 'Cidec protein_coding 10.43496017 0.002138853 ENSMUSG00000027160 'Ccdc34 protein_coding 10.41562348 0.016713908 ENSMUSG00000021453 'Gadd45g protein_coding 10.40987579 0.016816113 ENSMUSG00000036863 'Syde2 protein_coding 10.40438599 0.002091357 ENSMUSG00000020801 'Med31 protein_coding 10.37619307 0.01700488 ENSMUSG00000056019 'Zfp709 protein_coding 10.36174748 0.017166044 ENSMUSG00000050908 'Tvp23a protein_coding 10.30792469 0.002230677 ENSMUSG00000086968 '4933431E20Rik antisense 10.29825333 0.017812494 ENSMUSG00000078484 'Klhl17 protein_coding 10.26130366 0.018075202 ENSMUSG00000003190 'Bcl2l12 protein_coding 10.23506056 0.018298591 ENSMUSG00000072653 'Zfp783 transcribed_unprocessed_pseudogene 10.02535189 0.020165934 ENSMUSG00000032842 'Abcc10 protein_coding 10.01174838 0.020180687 ENSMUSG00000062110 'Scfd2 protein_coding 9.97540882 0.022242108 ENSMUSG00000024713 'Pcsk5 protein_coding 9.899685992 0.021293153 ENSMUSG00000031107 'Rbmx2 protein_coding 9.707595915 0.022889225 ENSMUSG00000107741 'Gm2011 transcribed_unprocessed_pseudogene 9.702328161 0.003483051 ENSMUSG00000092412 'Gm20507 processed_transcript 9.685851216 0.023909494 ENSMUSG00000080152 'Gm12260 unprocessed_pseudogene 9.518960132 0.024773698 ENSMUSG00000075511 '1700001L05Rik lincRNA 9.513427109 0.003841741 ENSMUSG00000091705 'H2-Q2 protein_coding 9.273260636 0.027002157 ENSMUSG00000104292 'Gm38042 TEC 9.245759347 0.030488343 ENSMUSG00000046826 'Fam187b protein_coding 9.202007921 0.02782233 ENSMUSG00000013155 'Enkd1 protein_coding 9.030536732 0.029959131 ENSMUSG00000047246 'Hist1h2be protein_coding 8.875530825 0.031745904 ENSMUSG00000089698 'Gm2541 processed_pseudogene 8.719421945 0.033782652 ENSMUSG00000097322 'A530083I20Rik lincRNA 8.698370879 0.034331441 ENSMUSG00000033594 'Spata2l protein_coding 8.698291497 0.006743172 ENSMUSG00000058331 'Zfp85 protein_coding 8.650391669 0.034710902 ENSMUSG00000110612 'RP24-93F18.4 processed_pseudogene 8.642467968 0.037712024 ENSMUSG00000048997 'Atxn7l2 protein_coding 8.636548281 0.034706324 ENSMUSG00000053040 'Aph1c protein_coding 8.477592064 0.037363905 ENSMUSG00000021066 'Atl1 protein_coding 8.275244162 0.039997129 ENSMUSG00000102018 'K230010J24Rik protein_coding 8.067324095 0.044279813 ENSMUSG00000085154 'C130046K22Rik processed_transcript 8.057465197 0.010141845 ENSMUSG00000085287 '4833418N02Rik lincRNA 7.827839376 0.048360657 ENSMUSG00000102982 'Gm38319 TEC 7.565158963 0.04211604 ENSMUSG00000095199 'Zfp967 protein_coding 7.499809008 0.015669301 ENSMUSG00000095545 'Zfp969 protein_coding 7.499809008 0.015670416 ENSMUSG00000075389 '2810410L24Rik processed_transcript 7.33409643 0.016700931 ENSMUSG00000053574 '4930563E22Rik processed_transcript 6.18657728 0.037462846 ENSMUSG00000086455 'Gm11815 lincRNA 6.133252608 0.038768101 ENSMUSG00000083327 'Vcp-rs processed_pseudogene -5.273551697 0.045006112 ENSMUSG00000003153 'Slc2a3 protein_coding -7.275959025 0.017594658 ENSMUSG00000104156 'Gm38102 lincRNA -7.992579054 0.046038638 ENSMUSG00000079429 'Mroh2a protein_coding -8.073188007 0.043723138 ENSMUSG00000031425 'Plp1 protein_coding -8.284556152 0.040621869 ENSMUSG00000110684 'RP23-91H23.3 lincRNA -8.304599092 0.041023168 ENSMUSG00000084803 '5830444B04Rik processed_transcript -8.348027921 0.039783283 ENSMUSG00000037375 'Hhat protein_coding -8.368327576 0.03933721 ENSMUSG00000047534 'Mis18bp1 protein_coding -8.427358009 0.038713078 ENSMUSG00000106446 'Gm42970 TEC -8.435663536 0.013247411 ENSMUSG00000045744 'Bricd5 protein_coding -8.505906491 0.037067424 ENSMUSG00000108763 'Gm36028 protein_coding -8.571621949 0.036109905 ENSMUSG00000072109 'A530040E14Rik antisense -8.678827244 0.007311091 ENSMUSG00000025226 'Fbxl15 protein_coding -8.790939418 0.033436763 ENSMUSG00000020844 'Nxn protein_coding -8.822185423 0.032959285 ENSMUSG00000086370 'Ftx lincRNA -8.853046286 0.035116031 ENSMUSG00000060206 'Zfp462 protein_coding -8.885090297 0.032256872 ENSMUSG00000093938 'Evi2b protein_coding -9.096885753 0.029185505 ENSMUSG00000033233 'Trim45 protein_coding -9.217308371 0.027805846 ENSMUSG00000059327 'Eda protein_coding -9.21837864 0.027835915 ENSMUSG00000011305 'Plin5 protein_coding -9.229161948 0.028346855 ENSMUSG00000055799 'Tcf7l1 protein_coding -9.240442304 0.028051777 ENSMUSG00000054893 'Zfp667 protein_coding -9.260499485 0.027540542 ENSMUSG00000040329 'Il7 protein_coding -9.321870841 0.029610466 ENSMUSG00000090397 'Gm17096 antisense -9.383284184 0.029736833 ENSMUSG00000028329 'Xpa protein_coding -9.592369656 0.024310998 ENSMUSG00000046080 'Clec9a protein_coding -9.596851489 0.033248503 ENSMUSG00000105552 'Gm19710 lincRNA -9.607106227 0.024102628 ENSMUSG00000036019 'Tmtc2 protein_coding -9.63038608 0.024118011 ENSMUSG00000069727 'Zfp975 protein_coding -9.631095236 0.023919464 ENSMUSG00000072621 'Slfn10-ps transcribed_unprocessed_pseudogene -9.71813688 0.023994496 Diabetes Page 44 of 62
ENSMUSG00000058099 'Nfam1 protein_coding -9.782770456 0.024827285 ENSMUSG00000078931 'Pdf protein_coding -9.895943493 0.004338979 ENSMUSG00000020657 'Dnajc27 protein_coding -10.0220107 0.020403329 ENSMUSG00000102752 'Gm7694 protein_coding -10.03266984 0.021863655 ENSMUSG00000066894 'Vsig10 protein_coding -10.10241999 0.019456397 ENSMUSG00000092592 'Gm20449 protein_coding -10.13093942 0.019321616 ENSMUSG00000024529 'Lox protein_coding -10.19621725 0.018987176 ENSMUSG00000027845 'Dclre1b protein_coding -10.26869658 0.018135954 ENSMUSG00000040723 'Rcsd1 protein_coding -10.2812241 0.00422781 ENSMUSG00000105748 'Gm43088 TEC -10.29959767 0.019453834 ENSMUSG00000041945 'Mfsd9 protein_coding -10.41130778 0.016885758 ENSMUSG00000078630 'Tomt protein_coding -10.43940516 0.002030626 ENSMUSG00000037243 'Zfp692 protein_coding -10.48312052 0.016405617 ENSMUSG00000012126 'Ubxn11 protein_coding -10.48932501 0.01631612 ENSMUSG00000026335 'Pam protein_coding -10.54791201 0.016075235 ENSMUSG00000107624 'Gm44005 TEC -10.55794767 0.015995112 ENSMUSG00000034748 'Sirt6 protein_coding -10.58280529 0.015632644 ENSMUSG00000083392 'Gm6335 processed_pseudogene -10.60775527 0.015376101 ENSMUSG00000108500 'Gm45033 TEC -10.68517171 0.015332631 ENSMUSG00000002486 'Tchp protein_coding -10.8085825 0.001559005 ENSMUSG00000024691 'Fam111a protein_coding -10.81640261 0.014723765 ENSMUSG00000002059 'Rab34 protein_coding -10.8180089 0.013957594 ENSMUSG00000041219 'Arhgap11a protein_coding -10.87215389 0.013539771 ENSMUSG00000028696 'Ipp protein_coding -10.87978057 0.013469066 ENSMUSG00000028884 'Rpa2 protein_coding -10.88952888 0.013467595 ENSMUSG00000102813 'Gm37795 TEC -10.91210728 0.013363946 ENSMUSG00000038007 'Acer2 protein_coding -10.92781535 0.013155169 ENSMUSG00000078234 'Klhdc7a protein_coding -10.94445167 0.001595642 ENSMUSG00000020709 'Adap2 protein_coding -10.99250412 0.013212306 ENSMUSG00000104291 'A130071D04Rik TEC -11.00902779 0.049970327 ENSMUSG00000029428 'Stx2 protein_coding -11.0094029 0.049980076 ENSMUSG00000025147 'Mob2 protein_coding -11.01578631 0.049893236 ENSMUSG00000003531 'Dgcr6 protein_coding -11.01685525 0.049854187 ENSMUSG00000041836 'Ptpre protein_coding -11.02426931 0.049760026 ENSMUSG00000104406 'Gm38014 TEC -11.02680844 0.049722729 ENSMUSG00000005057 'Sh2b2 protein_coding -11.02721099 0.013045383 ENSMUSG00000025477 'Inpp5a protein_coding -11.03658446 0.049586449 ENSMUSG00000028420 'Tmem38b protein_coding -11.04628558 0.049485314 ENSMUSG00000072964 'Bhlhb9 protein_coding -11.04857968 0.04940813 ENSMUSG00000028480 'Glipr2 protein_coding -11.0499225 0.049372659 ENSMUSG00000104449 'Gm37255 TEC -11.05173754 0.049333357 ENSMUSG00000014245 'Pigl protein_coding -11.05436079 0.012471484 ENSMUSG00000002550 'Uck1 protein_coding -11.05716898 0.049271583 ENSMUSG00000025779 'Ly96 protein_coding -11.06039415 0.012413291 ENSMUSG00000035104 'Eva1a protein_coding -11.06047599 0.049196047 ENSMUSG00000108478 'Gm19514 TEC -11.06172552 0.049168596 ENSMUSG00000042284 'Itga1 protein_coding -11.0651712 0.049122266 ENSMUSG00000042138 'Msantd2 protein_coding -11.06655488 0.012400027 ENSMUSG00000037315 'Jade3 protein_coding -11.07887219 0.048816769 ENSMUSG00000032478 'Nme6 protein_coding -11.0788722 0.048810819 ENSMUSG00000029587 'Zfp12 protein_coding -11.0793272 0.012368125 ENSMUSG00000053318 'Slamf8 protein_coding -11.08064419 0.048771399 ENSMUSG00000039967 'Zfp292 protein_coding -11.08224876 0.048753167 ENSMUSG00000015335 'Zdhhc12 protein_coding -11.08567421 0.048674612 ENSMUSG00000031381 'Piga protein_coding -11.0858266 0.012347805 ENSMUSG00000098905 'Zfp953 protein_coding -11.0939347 0.048541058 ENSMUSG00000026657 'Frmd4a protein_coding -11.09393471 0.048524682 ENSMUSG00000026816 'Gtf3c5 protein_coding -11.09581746 0.048524174 ENSMUSG00000020056 'Ccdc53 protein_coding -11.09581746 0.048529201 ENSMUSG00000025255 'Zfhx4 protein_coding -11.09723829 0.048499092 ENSMUSG00000038214 'Bend3 protein_coding -11.10053433 0.048426604 ENSMUSG00000020231 'Dip2a protein_coding -11.10254023 0.048350901 ENSMUSG00000046792 'Zfp787 protein_coding -11.10340408 0.048351277 ENSMUSG00000001999 'Blvra protein_coding -11.10689999 0.048229462 ENSMUSG00000022672 'Prkdc protein_coding -11.10710392 0.048213561 ENSMUSG00000043384 'Gprasp1 protein_coding -11.10923182 0.048149345 ENSMUSG00000009292 'Trpm2 protein_coding -11.11527404 0.048032713 ENSMUSG00000021712 'Trim23 protein_coding -11.11733721 0.047996853 ENSMUSG00000028859 'Csf3r protein_coding -11.12339814 0.047903395 ENSMUSG00000038280 'Ostm1 protein_coding -11.12425365 0.047885903 ENSMUSG00000108181 'C030015A19Rik TEC -11.12582625 0.04784605 ENSMUSG00000109585 'RP23-383A10.1 TEC -11.12582625 0.047851079 ENSMUSG00000102664 'Gm38380 TEC -11.12747524 0.04781692 ENSMUSG00000026696 'Vamp4 protein_coding -11.12986466 0.047777644 ENSMUSG00000019368 'Sec14l4 protein_coding -11.13632982 0.012226688 ENSMUSG00000016283 'H2-M2 protein_coding -11.1395104 0.047589981 ENSMUSG00000022438 'Parvb protein_coding -11.1395104 0.047595748 ENSMUSG00000041623 'D11Wsu47e protein_coding -11.14028063 0.011909099 ENSMUSG00000103970 'Gm37678 TEC -11.14404199 0.011975993 ENSMUSG00000026222 'Sp100 protein_coding -11.15666136 0.047291105 ENSMUSG00000031434 'Morc4 protein_coding -11.15834702 0.047227699 ENSMUSG00000032754 'Slc8b1 protein_coding -11.1616757 0.047147866 ENSMUSG00000028102 'Pex11b protein_coding -11.16176946 0.047135802 Page 45 of 62 Diabetes
ENSMUSG00000003031 'Cdkn1b protein_coding -11.17130017 0.04695441 ENSMUSG00000058291 'Zfp68 protein_coding -11.177681 0.046827522 ENSMUSG00000099931 'Gm29358 sense_intronic -11.17842289 0.046811695 ENSMUSG00000040472 'Rabggta protein_coding -11.18679484 0.046640751 ENSMUSG00000104348 'Gm37691 TEC -11.19665589 0.046511812 ENSMUSG00000024742 'Fen1 protein_coding -11.20528303 0.046381908 ENSMUSG00000067224 'Gm3695 processed_pseudogene -11.20639385 0.046333536 ENSMUSG00000035095 'Fam167a protein_coding -11.2083415 0.046298252 ENSMUSG00000103928 'Gm37893 TEC -11.21383298 0.046186566 ENSMUSG00000026272 'Agxt protein_coding -11.21451845 0.046196372 ENSMUSG00000091736 'Yy2 protein_coding -11.2261982 0.045932739 ENSMUSG00000073421 'H2-Ab1 protein_coding -11.22957111 0.045872447 ENSMUSG00000066640 'Fbxl18 protein_coding -11.23257857 0.045816469 ENSMUSG00000028619 'Tceanc2 protein_coding -11.23702685 0.045669527 ENSMUSG00000040363 'Bcor protein_coding -11.23998356 0.045582145 ENSMUSG00000031166 'Wdr13 protein_coding -11.24156352 0.045526556 ENSMUSG00000034320 'Slc26a2 protein_coding -11.24156353 0.045511214 ENSMUSG00000022677 'Fopnl protein_coding -11.24656682 0.045378766 ENSMUSG00000018199 'Trove2 protein_coding -11.24910116 0.045289203 ENSMUSG00000005374 'Tbl2 protein_coding -11.24973284 0.045273054 ENSMUSG00000021785 'Ngly1 protein_coding -11.25049287 0.045238273 ENSMUSG00000008845 'Cd163 protein_coding -11.26818741 0.044961755 ENSMUSG00000101823 'Gm29438 TEC -11.2701541 0.044935118 ENSMUSG00000089847 'Timm10b protein_coding -11.27170263 0.044927004 ENSMUSG00000033933 'Vhl protein_coding -11.27403769 0.044888256 ENSMUSG00000052934 'Fbxo31 protein_coding -11.27410261 0.011015144 ENSMUSG00000038451 'Spsb2 protein_coding -11.27886264 0.010980911 ENSMUSG00000042302 'Ehbp1 protein_coding -11.27907551 0.011040952 ENSMUSG00000035459 'Stab2 protein_coding -11.28276888 0.044698842 ENSMUSG00000105453 'Gm43702 TEC -11.2868748 0.010998738 ENSMUSG00000042677 'Zc3h12a protein_coding -11.30262882 0.044365259 ENSMUSG00000032265 'Fam46a protein_coding -11.30554514 0.044287115 ENSMUSG00000022553 'Maf1 protein_coding -11.30556451 0.044302949 ENSMUSG00000042015 'Wdr41 protein_coding -11.30860382 0.044246504 ENSMUSG00000018548 'Trim37 protein_coding -11.32579596 0.043916405 ENSMUSG00000096740 'Lbhd1 protein_coding -11.32810831 0.013010828 ENSMUSG00000035392 'Dennd1a protein_coding -11.33127162 0.043847177 ENSMUSG00000021639 'Gtf2h2 protein_coding -11.33687209 0.043731564 ENSMUSG00000075591 'Gm10874 lincRNA -11.3424509 0.043599265 ENSMUSG00000020277 'Pfkl protein_coding -11.34754291 0.043482073 ENSMUSG00000084143 'Gm13400 processed_pseudogene -11.34800823 0.043469197 ENSMUSG00000106262 'Gm43375 TEC -11.35142189 0.04339244 ENSMUSG00000028882 'Ppp1r8 protein_coding -11.35344135 0.043363894 ENSMUSG00000078440 'Dohh protein_coding -11.36224402 0.043164242 ENSMUSG00000033773 'Rpap2 protein_coding -11.36546427 0.043120743 ENSMUSG00000039201 'Tbc1d25 protein_coding -11.37367198 0.010390741 ENSMUSG00000073609 'D2hgdh protein_coding -11.37649183 0.010416785 ENSMUSG00000031832 'Taf1c protein_coding -11.37660058 0.042914985 ENSMUSG00000020079 'Supv3l1 protein_coding -11.37968987 0.04284881 ENSMUSG00000048440 'Cyp4f16 protein_coding -11.38901949 0.042676511 ENSMUSG00000028861 'Mrps15 protein_coding -11.39120433 0.042640444 ENSMUSG00000038145 'Snrk protein_coding -11.39574277 0.042585603 ENSMUSG00000066621 'Tecpr1 protein_coding -11.39978026 0.042500918 ENSMUSG00000001751 'Naglu protein_coding -11.40134736 0.042484055 ENSMUSG00000042659 'Arrdc4 protein_coding -11.40243487 0.042412975 ENSMUSG00000031520 'Vegfc protein_coding -11.40592297 0.010264786 ENSMUSG00000053553 '3110082I17Rik protein_coding -11.42497937 0.010114196 ENSMUSG00000092329 'Gm20388 protein_coding -11.42835493 0.041900833 ENSMUSG00000060862 'Zbtb40 protein_coding -11.43079625 0.041862328 ENSMUSG00000044475 'Ascc1 protein_coding -11.4327462 0.010096816 ENSMUSG00000027274 'Mkks protein_coding -11.4335831 0.041818693 ENSMUSG00000009995 'Taz protein_coding -11.43413178 0.041806804 ENSMUSG00000021930 'Spryd7 protein_coding -11.44432007 0.04157426 ENSMUSG00000036916 'Zfp280c protein_coding -11.44467697 0.041562845 ENSMUSG00000028057 'Rit1 protein_coding -11.44941127 0.010337594 ENSMUSG00000044700 'Tmem201 protein_coding -11.45019207 0.041429219 ENSMUSG00000026470 'Stx6 protein_coding -11.45230507 0.041384106 ENSMUSG00000082286 'Pisd-ps1 transcribed_unprocessed_pseudogene -11.45488267 0.041325448 ENSMUSG00000028763 'Hspg2 protein_coding -11.45745567 0.041260135 ENSMUSG00000108042 'B130021K23Rik TEC -11.46011047 0.041149457 ENSMUSG00000017299 'Dnttip1 protein_coding -11.46258795 0.041120735 ENSMUSG00000049988 'Lrrc25 protein_coding -11.47025228 0.040995439 ENSMUSG00000041642 'Kif21b protein_coding -11.48040847 0.040783552 ENSMUSG00000043633 'Fam221b protein_coding -11.48089904 0.040763554 ENSMUSG00000055413 'H2-Q5 polymorphic_pseudogene -11.4815012 0.040753636 ENSMUSG00000029370 'Rassf6 protein_coding -11.48604972 0.040586289 ENSMUSG00000018669 'Cdk5rap3 protein_coding -11.48821853 0.040516952 ENSMUSG00000024777 'Ppp2r5b protein_coding -11.49089672 0.040386213 ENSMUSG00000028958 'Tmub1 protein_coding -11.49446419 0.000896491 ENSMUSG00000040413 'Timd2 protein_coding -11.4960689 0.009544981 ENSMUSG00000017830 'Dhx58 protein_coding -11.50300184 0.040136137 ENSMUSG00000008136 'Fhl2 protein_coding -11.50320255 0.009584534 ENSMUSG00000020659 'Cbll1 protein_coding -11.50549051 0.040055511 Diabetes Page 46 of 62
ENSMUSG00000019139 'Isyna1 protein_coding -11.50549051 0.040069011 ENSMUSG00000041654 'Slc39a11 protein_coding -11.50797489 0.039993372 ENSMUSG00000051579 'Tceal8 protein_coding -11.51214523 0.039882866 ENSMUSG00000028857 'Tmem222 protein_coding -11.51293087 0.039850963 ENSMUSG00000028920 'Fbxo42 protein_coding -11.51657346 0.039799796 ENSMUSG00000034333 'Zbed4 protein_coding -11.5166367 0.039795601 ENSMUSG00000031352 'Hccs protein_coding -11.51740882 0.039788524 ENSMUSG00000033767 'D930015E06Rik protein_coding -11.52279201 0.039706404 ENSMUSG00000069805 'Fbp1 protein_coding -11.53308534 0.039527424 ENSMUSG00000019762 'Iyd protein_coding -11.53827322 0.039412405 ENSMUSG00000024208 'Uqcc2 protein_coding -11.53827322 0.03940805 ENSMUSG00000002679 'Med6 protein_coding -11.54231438 0.039342313 ENSMUSG00000043262 'Uevld protein_coding -11.54523157 0.039292959 ENSMUSG00000050002 'Idnk protein_coding -11.55116188 0.039170567 ENSMUSG00000030726 'Pold3 protein_coding -11.55873896 0.039008833 ENSMUSG00000010914 'Pdhx protein_coding -11.56139054 0.038962416 ENSMUSG00000026571 'Dcaf6 protein_coding -11.56361955 0.038924841 ENSMUSG00000038058 'Nod1 protein_coding -11.56832267 0.009300202 ENSMUSG00000033308 'Dpyd protein_coding -11.56848369 0.038745581 ENSMUSG00000020910 'Adprm protein_coding -11.56848369 0.03875936 ENSMUSG00000027381 'Bcl2l11 protein_coding -11.56864643 0.009122433 ENSMUSG00000039738 'Slx4 protein_coding -11.56873346 0.038719471 ENSMUSG00000079043 'Fastkd5 protein_coding -11.57090962 0.038647031 ENSMUSG00000019916 'P4ha1 protein_coding -11.57111139 0.038634375 ENSMUSG00000007036 'Abhd16a protein_coding -11.5765988 0.038509971 ENSMUSG00000038578 'Susd1 protein_coding -11.579118 0.038458288 ENSMUSG00000038116 'Phf20 protein_coding -11.58764795 0.038338814 ENSMUSG00000072692 'Rpl37rt transcribed_processed_pseudogene -11.59662936 0.008878532 ENSMUSG00000060376 'Bckdha protein_coding -11.59970701 0.038095549 ENSMUSG00000074794 'Arrdc3 protein_coding -11.60697318 0.009068725 ENSMUSG00000027667 'Zfp639 protein_coding -11.6114735 0.037895888 ENSMUSG00000024309 'Pfdn6 protein_coding -11.6162438 0.037768605 ENSMUSG00000106706 'C530043K16Rik TEC -11.6162438 0.037782038 ENSMUSG00000089943 'Ugt1a5 protein_coding -11.61661004 0.008788686 ENSMUSG00000018927 'Ccl6 protein_coding -11.61786472 0.037694616 ENSMUSG00000057363 'Uxs1 protein_coding -11.6185908 0.037673087 ENSMUSG00000029007 'Agtrap protein_coding -11.62016309 0.037647678 ENSMUSG00000030649 'Anapc15 protein_coding -11.621705 0.000769994 ENSMUSG00000110471 'RP23-212H13.6 sense_intronic -11.62817864 0.037513204 ENSMUSG00000051329 'Nup160 protein_coding -11.63259319 0.037419281 ENSMUSG00000002010 'Idh3g protein_coding -11.64912616 0.037139353 ENSMUSG00000025001 'Hells protein_coding -11.65431477 0.008523758 ENSMUSG00000104903 'Gm43707 sense_intronic -11.65905706 0.036968115 ENSMUSG00000028944 'Prkag2 protein_coding -11.66247335 0.036888416 ENSMUSG00000035847 'Ids protein_coding -11.66462814 0.036834882 ENSMUSG00000028266 'Lmo4 protein_coding -11.66537775 0.036784053 ENSMUSG00000069206 'Zfp874a protein_coding -11.67041339 0.036658588 ENSMUSG00000058392 'Rrp1b protein_coding -11.67154412 0.036610598 ENSMUSG00000026788 'Zbtb43 protein_coding -11.67154412 0.036623639 ENSMUSG00000021417 'Eci2 protein_coding -11.68417303 0.03638642 ENSMUSG00000035790 'Cep19 protein_coding -11.68831053 0.000718838 ENSMUSG00000038473 'Nos1ap protein_coding -11.69301489 0.008524109 ENSMUSG00000101970 '1810026B05Rik processed_transcript -11.69620549 0.036050365 ENSMUSG00000020153 'Ndufs7 protein_coding -11.69620549 0.036054768 ENSMUSG00000013822 'Elof1 protein_coding -11.70670717 0.035831253 ENSMUSG00000002983 'Relb protein_coding -11.70726742 0.035794029 ENSMUSG00000036820 'Amdhd2 protein_coding -11.70921836 0.035774881 ENSMUSG00000032280 'Tle3 protein_coding -11.712136 0.008221976 ENSMUSG00000039623 'Ap5z1 protein_coding -11.7253211 0.035415698 ENSMUSG00000105848 'Gm42683 TEC -11.72697975 0.035371792 ENSMUSG00000021466 'Ptch1 protein_coding -11.73409314 0.035249148 ENSMUSG00000025269 'Apex2 protein_coding -11.74291005 0.00794849 ENSMUSG00000032411 'Tfdp2 protein_coding -11.74418432 0.008034305 ENSMUSG00000029625 'Cpsf4 protein_coding -11.74756717 0.035043785 ENSMUSG00000040111 'Gramd1b protein_coding -11.74756717 0.035056278 ENSMUSG00000066861 'Oas1g protein_coding -11.74871298 0.035019837 ENSMUSG00000026421 'Csrp1 protein_coding -11.74966807 0.034993789 ENSMUSG00000012117 'Dhdds protein_coding -11.74966807 0.035006226 ENSMUSG00000028648 'Ndufs5 protein_coding -11.7611686 0.03483289 ENSMUSG00000015016 'Acsf3 protein_coding -11.76122479 0.007952286 ENSMUSG00000022897 'Dyrk1a protein_coding -11.76773065 0.034727902 ENSMUSG00000028898 'Trnau1ap protein_coding -11.76843968 0.034705923 ENSMUSG00000052926 'Rnaseh2a protein_coding -11.77051041 0.034685503 ENSMUSG00000060950 'Trmt61a protein_coding -11.77195401 0.034654734 ENSMUSG00000037280 'Galnt6 protein_coding -11.78697017 0.034459706 ENSMUSG00000024300 'Myo1f protein_coding -11.79105594 0.034399619 ENSMUSG00000056938 'Acbd4 protein_coding -11.80056047 0.00788915 ENSMUSG00000042682 'Selk protein_coding -11.80122001 0.03423527 ENSMUSG00000072872 'Rybp protein_coding -11.80682953 0.034089196 ENSMUSG00000108389 'Gm45206 TEC -11.80694387 0.007606508 ENSMUSG00000094870 'Zfp131 protein_coding -11.80736203 0.03405441 ENSMUSG00000003062 'Stard3nl protein_coding -11.80897741 0.03401347 ENSMUSG00000037808 'Fam76b protein_coding -11.80972411 0.007604575 Page 47 of 62 Diabetes
ENSMUSG00000040464 'Gtpbp10 protein_coding -11.81332314 0.033947905 ENSMUSG00000000976 'Heatr6 protein_coding -11.81753715 0.033902453 ENSMUSG00000034709 'Ppp1r21 protein_coding -11.81966917 0.033859337 ENSMUSG00000020415 'Pttg1 protein_coding -11.81966917 0.033847845 ENSMUSG00000038342 'Mlxip protein_coding -11.83128954 0.033626814 ENSMUSG00000105519 'E430021H15Rik TEC -11.83661285 0.033527577 ENSMUSG00000017652 'Cd40 protein_coding -11.83991164 0.033443096 ENSMUSG00000010047 'Hyal2 protein_coding -11.84593377 0.033319651 ENSMUSG00000018999 'Slc35b4 protein_coding -11.84993461 0.033238871 ENSMUSG00000006378 'Gcat protein_coding -11.85176251 0.007381454 ENSMUSG00000047804 'Akap10 protein_coding -11.85880001 0.033078305 ENSMUSG00000020262 'Adarb1 protein_coding -11.85988843 0.033028718 ENSMUSG00000028556 'Dock7 protein_coding -11.86681546 0.032923598 ENSMUSG00000023079 'Gtf2ird1 protein_coding -11.87139657 0.03285533 ENSMUSG00000039556 'Ppp1r3f protein_coding -11.88388409 0.032639372 ENSMUSG00000040795 'Iqcc protein_coding -11.8881112 0.007168442 ENSMUSG00000033581 'Igf2bp2 protein_coding -11.88886804 0.007231606 ENSMUSG00000063362 'Alg11 protein_coding -11.88961131 0.032527542 ENSMUSG00000029213 'Commd8 protein_coding -11.89341685 0.032485761 ENSMUSG00000064032 'Gm10143 processed_transcript -11.89355383 0.007136152 ENSMUSG00000028217 'Cdh17 protein_coding -11.89439607 0.032441033 ENSMUSG00000039382 'Wdr45 protein_coding -11.89531588 0.032421841 ENSMUSG00000031633 'Slc25a4 protein_coding -11.9044753 0.032286269 ENSMUSG00000053819 'Camk2d protein_coding -11.90541889 0.007445663 ENSMUSG00000048865 'Arhgap30 protein_coding -11.90973311 0.001042519 ENSMUSG00000063894 'Zkscan8 protein_coding -11.91589343 0.00700473 ENSMUSG00000034998 'Foxn2 protein_coding -11.91791123 0.032031051 ENSMUSG00000003868 'Ruvbl2 protein_coding -11.92244235 0.03190996 ENSMUSG00000027411 'Vps16 protein_coding -11.92391547 0.031871649 ENSMUSG00000078153 'Psme2b protein_coding -11.92391547 0.031883054 ENSMUSG00000033411 'Ctdspl2 protein_coding -11.9242403 0.006966076 ENSMUSG00000067365 'Tmem128 protein_coding -11.92907758 0.031764933 ENSMUSG00000028617 'Lrrc42 protein_coding -11.93278057 0.031712565 ENSMUSG00000099881 '2810013P06Rik bidirectional_promoter_lncRNA -11.93525751 0.031668108 ENSMUSG00000026933 'Camsap1 protein_coding -11.94107771 0.031551117 ENSMUSG00000019261 'Map1s protein_coding -11.94199668 0.03152969 ENSMUSG00000036555 'Iqce protein_coding -11.96712094 0.006754465 ENSMUSG00000025041 'Nt5c2 protein_coding -11.97312454 0.031069833 ENSMUSG00000036432 'Siah2 protein_coding -11.97852969 0.006671085 ENSMUSG00000026858 'Fam73b protein_coding -11.98980034 0.00662969 ENSMUSG00000016024 'Lbp protein_coding -11.99404237 0.030758878 ENSMUSG00000078713 'Tomm5 protein_coding -11.99781323 0.030687724 ENSMUSG00000024044 'Epb41l3 protein_coding -11.99874231 0.03064838 ENSMUSG00000005469 'Prkaca protein_coding -11.99962528 0.03058742 ENSMUSG00000033096 'Apmap protein_coding -11.99969497 0.030599186 ENSMUSG00000042729 'Wdr74 protein_coding -12.00329597 0.030494657 ENSMUSG00000040236 'Trappc5 protein_coding -12.00719749 0.030428827 ENSMUSG00000027261 'Hao1 protein_coding -12.00824549 0.006783552 ENSMUSG00000031444 'F10 protein_coding -12.01279889 0.030323841 ENSMUSG00000025959 'Klf7 protein_coding -12.01717215 0.030246743 ENSMUSG00000024325 'Ring1 protein_coding -12.02134076 0.006438915 ENSMUSG00000036282 'Naa30 protein_coding -12.02822598 0.030095806 ENSMUSG00000032571 'Pik3r4 protein_coding -12.03702618 0.029902763 ENSMUSG00000039985 'Fam60a protein_coding -12.04166088 0.006323419 ENSMUSG00000017405 'Nek8 protein_coding -12.04473297 0.000475922 ENSMUSG00000041625 'Ggact protein_coding -12.04953422 0.000467122 ENSMUSG00000023147 'Wrb protein_coding -12.04965199 0.006286724 ENSMUSG00000003808 'Farsa protein_coding -12.04993443 0.029592144 ENSMUSG00000025340 'Rabgef1 protein_coding -12.05322449 0.029544677 ENSMUSG00000032727 'Mier3 protein_coding -12.05574282 0.006237349 ENSMUSG00000021196 'Pfkp protein_coding -12.05704357 0.006568494 ENSMUSG00000026810 'Dpm2 protein_coding -12.05793019 0.029422605 ENSMUSG00000091831 'Gm4707 protein_coding -12.06350955 0.02932406 ENSMUSG00000022779 'Top3b protein_coding -12.07659052 0.029114524 ENSMUSG00000021147 'Wdr37 protein_coding -12.07695813 0.029072288 ENSMUSG00000035473 'Galm protein_coding -12.08149253 0.006084491 ENSMUSG00000001855 'Nup214 protein_coding -12.08547764 0.028898575 ENSMUSG00000028261 'Ndufaf4 protein_coding -12.09028251 0.028739287 ENSMUSG00000039680 'Mrps6 protein_coding -12.09737225 0.007242975 ENSMUSG00000034274 'Thoc5 protein_coding -12.09782832 0.028546223 ENSMUSG00000001665 'Gstt3 protein_coding -12.09885091 0.028521619 ENSMUSG00000021557 'Agtpbp1 protein_coding -12.09899391 0.005956272 ENSMUSG00000036534 'Slc38a7 protein_coding -12.10308929 0.028440618 ENSMUSG00000105561 'Gm43462 sense_intronic -12.10329171 0.006867651 ENSMUSG00000028399 'Ptprd protein_coding -12.1072319 0.028357525 ENSMUSG00000005410 'Mcm5 protein_coding -12.10917858 0.00592191 ENSMUSG00000079478 'Sssca1 protein_coding -12.11819468 0.028195618 ENSMUSG00000049130 'C5ar1 protein_coding -12.11981988 0.028145075 ENSMUSG00000027185 'Nat10 protein_coding -12.11981988 0.028155819 ENSMUSG00000040855 'Reps2 protein_coding -12.12071903 0.005844745 ENSMUSG00000022378 'Fam49b protein_coding -12.12630242 0.027977302 ENSMUSG00000041324 'Inhba protein_coding -12.13114528 0.027877368 ENSMUSG00000026589 'Sec16b protein_coding -12.13425844 0.027818583 Diabetes Page 48 of 62
ENSMUSG00000028016 'Ints12 protein_coding -12.14876462 0.027580694 ENSMUSG00000027751 'Supt20 protein_coding -12.15466994 0.027399397 ENSMUSG00000078619 'Smarcd2 protein_coding -12.15490433 0.027389542 ENSMUSG00000036167 'Pphln1 protein_coding -12.15974723 0.027315873 ENSMUSG00000034032 'Rpap1 protein_coding -12.15998472 0.005604428 ENSMUSG00000037049 'Smpd1 protein_coding -12.1614101 0.027244918 ENSMUSG00000109719 'RP24-247M18.5 TEC -12.16457393 0.02715996 ENSMUSG00000025324 'Atp10a protein_coding -12.17146166 0.027021293 ENSMUSG00000028126 'Pip5k1a protein_coding -12.17479664 0.026926268 ENSMUSG00000027207 'Galk2 protein_coding -12.1825404 0.00547833 ENSMUSG00000040760 'Appl1 protein_coding -12.19319985 0.026667326 ENSMUSG00000021948 'Prkcd protein_coding -12.19477365 0.026627709 ENSMUSG00000031583 'Wrn protein_coding -12.1954361 0.005495579 ENSMUSG00000001482 'Def8 protein_coding -12.19634573 0.02655827 ENSMUSG00000026821 'Ralgds protein_coding -12.1972918 0.026534129 ENSMUSG00000046785 'Epm2aip1 protein_coding -12.19773917 0.005400369 ENSMUSG00000073489 'Ifi204 protein_coding -12.20125038 0.005372035 ENSMUSG00000030956 'Fam53b protein_coding -12.20426912 0.006220867 ENSMUSG00000036817 'Sun1 protein_coding -12.20496814 0.026384534 ENSMUSG00000019738 'Polr2i protein_coding -12.20521067 0.000355344 ENSMUSG00000051147 'Nat2 protein_coding -12.20802727 0.02633666 ENSMUSG00000039599 'Fam149b protein_coding -12.21448582 0.005310815 ENSMUSG00000086841 '2410006H16Rik processed_transcript -12.22019934 0.026119328 ENSMUSG00000040883 'Tmem205 protein_coding -12.2228123 0.026030794 ENSMUSG00000074238 'Ap1ar protein_coding -12.23116415 0.006765443 ENSMUSG00000104814 'Gm42979 TEC -12.23203872 0.025847525 ENSMUSG00000061360 'Phf5a protein_coding -12.23226959 0.025827828 ENSMUSG00000003680 'Taf6l protein_coding -12.23307408 0.005185819 ENSMUSG00000023206 'Il15ra protein_coding -12.2351067 0.000365387 ENSMUSG00000072115 'Ang protein_coding -12.25260807 0.025526965 ENSMUSG00000026712 'Mrc1 protein_coding -12.2531525 0.025511732 ENSMUSG00000022844 'Pdia5 protein_coding -12.26361291 0.025367789 ENSMUSG00000028744 'Pqlc2 protein_coding -12.26420141 0.005051221 ENSMUSG00000022898 'Dscr3 protein_coding -12.27373745 0.025204231 ENSMUSG00000032204 'Aqp9 protein_coding -12.28073839 0.025119634 ENSMUSG00000040451 'Sgms1 protein_coding -12.28103667 0.00498612 ENSMUSG00000002944 'Cd36 protein_coding -12.28536974 0.025013621 ENSMUSG00000032661 'Oas3 protein_coding -12.29306681 0.024836843 ENSMUSG00000026888 'Grb14 protein_coding -12.2942943 0.005115796 ENSMUSG00000040339 'Fam102b protein_coding -12.29492693 0.004910119 ENSMUSG00000075486 'Commd6 protein_coding -12.29572336 0.004879205 ENSMUSG00000065979 'Cpped1 protein_coding -12.29941783 0.024713836 ENSMUSG00000103146 'Gm37745 TEC -12.30039806 0.004868585 ENSMUSG00000012296 'Tjap1 protein_coding -12.30352336 0.00485461 ENSMUSG00000017686 'Rhot1 protein_coding -12.30379983 0.024594425 ENSMUSG00000020520 'Galnt10 protein_coding -12.31829942 0.024380159 ENSMUSG00000028973 'Abcb8 protein_coding -12.31873711 0.004772033 ENSMUSG00000090553 'Snrpe protein_coding -12.31900701 0.024346717 ENSMUSG00000026558 'Uck2 protein_coding -12.32395038 0.024263584 ENSMUSG00000036199 'Ndufa13 protein_coding -12.32407499 0.024254247 ENSMUSG00000016946 'Kctd5 protein_coding -12.32681709 0.004728929 ENSMUSG00000030091 'Nup210 protein_coding -12.32817412 0.02417129 ENSMUSG00000027340 'Slc23a2 protein_coding -12.33391423 0.024076241 ENSMUSG00000037326 'Capn15 protein_coding -12.33476103 0.004685642 ENSMUSG00000030474 'Siglece protein_coding -12.33937734 0.023993529 ENSMUSG00000033931 'Rbm34 protein_coding -12.34199238 0.00464966 ENSMUSG00000031393 'Mecp2 protein_coding -12.34674303 0.00466009 ENSMUSG00000020873 'Slc35b1 protein_coding -12.3511433 0.023792503 ENSMUSG00000042271 'Nxt2 protein_coding -12.35734293 0.00027495 ENSMUSG00000015112 'Slc25a13 protein_coding -12.36098919 0.023672111 ENSMUSG00000110423 'RP23-62O7.9 protein_coding -12.39008968 0.02345224 ENSMUSG00000029440 'Psmd9 protein_coding -12.3930202 0.004512315 ENSMUSG00000031871 'Cdh5 protein_coding -12.3981479 0.023395145 ENSMUSG00000022529 'Zfp263 protein_coding -12.4010414 0.004580373 ENSMUSG00000029128 'Rab28 protein_coding -12.40658355 0.004483431 ENSMUSG00000011306 'Sugp1 protein_coding -12.40716208 0.004491209 ENSMUSG00000078681 'Tm2d3 protein_coding -12.41407209 0.005133391 ENSMUSG00000029102 'Hgfac protein_coding -12.41599769 0.02325451 ENSMUSG00000067995 'Gtf2f2 protein_coding -12.42091675 0.004474185 ENSMUSG00000029253 'Cenpc1 protein_coding -12.42129945 0.000262215 ENSMUSG00000034826 'Nup54 protein_coding -12.42144913 0.023211395 ENSMUSG00000022329 'Stk3 protein_coding -12.42378917 0.004467762 ENSMUSG00000104214 'Gm36638 TEC -12.42407101 0.023170198 ENSMUSG00000070733 'Fryl protein_coding -12.42495446 0.023164027 ENSMUSG00000019796 'Lrp11 protein_coding -12.42949108 0.004436111 ENSMUSG00000034345 'Gtf2h5 protein_coding -12.43054534 0.023121433 ENSMUSG00000006705 'Pknox1 protein_coding -12.4323757 0.004425441 ENSMUSG00000030123 'Plxnd1 protein_coding -12.43386304 0.023098956 ENSMUSG00000037434 'Slc30a1 protein_coding -12.43422065 0.004435049 ENSMUSG00000006519 'Cyba protein_coding -12.43647377 0.023092889 ENSMUSG00000022538 'Lsg1 protein_coding -12.43777736 0.023077987 ENSMUSG00000038848 'Ythdf1 protein_coding -12.44038102 0.023052591 ENSMUSG00000021621 'Zcchc9 protein_coding -12.44989444 0.004389165 Page 49 of 62 Diabetes
ENSMUSG00000028232 'Tmem68 protein_coding -12.45154252 0.00438483 ENSMUSG00000036676 'Tmtc3 protein_coding -12.45397366 0.02292228 ENSMUSG00000030284 'Creld1 protein_coding -12.45570098 0.004388196 ENSMUSG00000027624 'Epb41l1 protein_coding -12.46749669 0.004400958 ENSMUSG00000032422 'Snx14 protein_coding -12.47288231 0.022778239 ENSMUSG00000003809 'Gcdh protein_coding -12.47806163 0.02275492 ENSMUSG00000071713 'Csf2rb protein_coding -12.47824897 0.022752684 ENSMUSG00000034216 'Vps18 protein_coding -12.48014816 0.022748895 ENSMUSG00000001158 'Snrnp27 protein_coding -12.48738236 0.004330353 ENSMUSG00000089917 'Uckl1 protein_coding -12.48772001 0.022716818 ENSMUSG00000009090 'Ap1b1 protein_coding -12.48797752 0.022716128 ENSMUSG00000036327 'Qsox2 protein_coding -12.49133791 0.004322129 ENSMUSG00000020537 'Drg2 protein_coding -12.50662451 0.022601948 ENSMUSG00000049580 'Tsku protein_coding -12.5092688 0.022585555 ENSMUSG00000030595 'Nfkbib protein_coding -12.51143867 0.022569381 ENSMUSG00000055116 'Arntl protein_coding -12.51682332 0.000254268 ENSMUSG00000019794 'Katna1 protein_coding -12.52406011 0.004270952 ENSMUSG00000000386 'Mx1 protein_coding -12.5315517 0.022456336 ENSMUSG00000022471 'Xrcc6 protein_coding -12.5324303 0.004259692 ENSMUSG00000026358 'Rgs1 protein_coding -12.54085225 0.022401093 ENSMUSG00000026786 'Apbb1ip protein_coding -12.54931946 0.022341962 ENSMUSG00000030579 'Tyrobp protein_coding -12.55052501 0.022341615 ENSMUSG00000025626 'Phf6 protein_coding -12.56734357 0.004198091 ENSMUSG00000049354 'Dcaf7 protein_coding -12.56751888 0.022196644 ENSMUSG00000026181 'Ppm1f protein_coding -12.56848848 0.022173354 ENSMUSG00000025877 'Hk3 protein_coding -12.5732412 0.022135447 ENSMUSG00000026283 'Ing5 protein_coding -12.58056437 0.004166026 ENSMUSG00000035051 'Dhx57 protein_coding -12.58070118 0.000234901 ENSMUSG00000020782 'Llgl2 protein_coding -12.59112612 0.004169748 ENSMUSG00000004285 'Atp6v1f protein_coding -12.60384878 0.021936667 ENSMUSG00000031816 'Mthfsd protein_coding -12.60653218 0.004122694 ENSMUSG00000038909 'Kat7 protein_coding -12.61616718 0.02187806 ENSMUSG00000028902 'Sf3a3 protein_coding -12.61647713 0.021874272 ENSMUSG00000039886 'Tmem120a protein_coding -12.63003915 0.021830193 ENSMUSG00000025857 'Dnaaf5 protein_coding -12.63117388 0.004093526 ENSMUSG00000020831 '0610010K14Rik protein_coding -12.63136567 0.021823526 ENSMUSG00000020918 'Kat2a protein_coding -12.64738365 0.02175725 ENSMUSG00000019841 'Rev3l protein_coding -12.64865766 0.004075103 ENSMUSG00000028451 '1700022I11Rik protein_coding -12.65143224 0.004072288 ENSMUSG00000026260 'Ndufa10 protein_coding -12.65397523 0.021728892 ENSMUSG00000105466 'Gm42998 TEC -12.65997171 0.021707291 ENSMUSG00000061111 'Fam195b protein_coding -12.66175256 0.004062276 ENSMUSG00000021936 'Mapk8 protein_coding -12.66679397 0.004068967 ENSMUSG00000036246 'Gmip protein_coding -12.66699128 0.004111804 ENSMUSG00000025059 'Gk protein_coding -12.67402525 0.021653883 ENSMUSG00000001348 'Acp5 protein_coding -12.67505214 0.021637551 ENSMUSG00000066151 'Fkbp15 protein_coding -12.6799111 0.004035207 ENSMUSG00000026932 'Nacc2 protein_coding -12.69022119 0.004023726 ENSMUSG00000053113 'Socs3 protein_coding -12.69380045 0.021467406 ENSMUSG00000107756 'Gm44164 TEC -12.69653468 0.021448065 ENSMUSG00000008450 'Nutf2 protein_coding -12.69930921 0.021434528 ENSMUSG00000024862 'Klc2 protein_coding -12.69972468 0.003996763 ENSMUSG00000027573 'Gid8 protein_coding -12.70791468 0.004003807 ENSMUSG00000031060 'Rbm10 protein_coding -12.72014874 0.003984934 ENSMUSG00000102496 'Gm36989 TEC -12.72567663 0.021396173 ENSMUSG00000022149 'C9 protein_coding -12.73243899 0.003987931 ENSMUSG00000003848 'Nob1 protein_coding -12.73737991 0.003977861 ENSMUSG00000042190 'Cmklr1 protein_coding -12.74629875 0.021337267 ENSMUSG00000029759 'Pon3 protein_coding -12.7494306 0.02131481 ENSMUSG00000056144 'Trim34a protein_coding -12.75576799 0.021237538 ENSMUSG00000004085 'Zak protein_coding -12.75903652 0.02120076 ENSMUSG00000028108 'Ecm1 protein_coding -12.78024065 0.003911617 ENSMUSG00000053063 'Clec12a protein_coding -12.78266136 0.021095748 ENSMUSG00000038250 'Usp38 protein_coding -12.79671252 0.003900735 ENSMUSG00000037936 'Scarb1 protein_coding -12.80878496 0.02106296 ENSMUSG00000032939 'Nup93 protein_coding -12.8097287 0.021060221 ENSMUSG00000018858 'Ict1 protein_coding -12.81130246 0.003899579 ENSMUSG00000015950 'Ncf1 protein_coding -12.81311606 0.021066129 ENSMUSG00000030753 'Prkrir protein_coding -12.81313629 0.003899358 ENSMUSG00000059534 'Uqcr10 protein_coding -12.8133189 0.021065489 ENSMUSG00000040455 'Usp45 protein_coding -12.81468473 0.003898454 ENSMUSG00000028703 'Lrrc41 protein_coding -12.84272784 0.020876382 ENSMUSG00000005804 'Bloc1s6 protein_coding -12.86143148 0.003829256 ENSMUSG00000049553 'Polr1a protein_coding -12.86830004 0.020796622 ENSMUSG00000082988 'Gm5903 processed_pseudogene -12.87056435 0.020798849 ENSMUSG00000022051 'Bnip3l protein_coding -12.87691461 0.020778945 ENSMUSG00000005813 'Metap1 protein_coding -12.8799827 0.020772881 ENSMUSG00000008333 'Snrpb2 protein_coding -12.89228887 0.003836551 ENSMUSG00000039244 'E130309D02Rik protein_coding -12.8987076 0.00380618 ENSMUSG00000031358 'Msl3 protein_coding -12.90065599 0.003796681 ENSMUSG00000041815 'Poldip3 protein_coding -12.90174798 0.020678977 ENSMUSG00000044206 'Vsig4 protein_coding -12.90552169 0.020663333 ENSMUSG00000000134 'Tfe3 protein_coding -12.90558299 0.020660646 Diabetes Page 50 of 62
ENSMUSG00000003161 'Sri protein_coding -12.90740485 0.020654542 ENSMUSG00000050730 'Arhgap42 protein_coding -12.90750377 0.020655863 ENSMUSG00000045409 'Trim39 protein_coding -12.90753091 0.000201407 ENSMUSG00000105383 'Gm42641 protein_coding -12.90796631 0.003957601 ENSMUSG00000029009 'Mthfr protein_coding -12.91516141 0.020662653 ENSMUSG00000044792 'Isca1 protein_coding -12.91934864 0.003792064 ENSMUSG00000024993 'Fam45a protein_coding -12.92632479 0.00379734 ENSMUSG00000006611 'Hfe protein_coding -12.93106021 0.003796649 ENSMUSG00000029084 'Cd38 protein_coding -12.94922287 0.005154275 ENSMUSG00000021185 '9030617O03Rik protein_coding -12.9555705 0.003850844 ENSMUSG00000028843 'Sh3bgrl3 protein_coding -12.96389508 0.003768397 ENSMUSG00000037916 'Ndufv1 protein_coding -12.96443063 0.020576436 ENSMUSG00000030515 'Tarsl2 protein_coding -12.96937322 0.003797517 ENSMUSG00000046671 'Mtfr1l protein_coding -12.9824999 0.020526689 ENSMUSG00000068699 'Flnc protein_coding -12.985794 0.000197608 ENSMUSG00000019988 'Nedd1 protein_coding -12.98616328 0.003755643 ENSMUSG00000030846 'Tial1 protein_coding -12.9868595 0.020554637 ENSMUSG00000024387 'Csnk2b protein_coding -12.99501189 0.020534058 ENSMUSG00000109279 'Gm45220 TEC -12.99636615 0.020525779 ENSMUSG00000003363 'Pld3 protein_coding -13.00473022 0.020533022 ENSMUSG00000050856 'Atp5k protein_coding -13.00530076 0.003758849 ENSMUSG00000027860 'Vangl1 protein_coding -13.02331694 0.003749665 ENSMUSG00000050608 'Minos1 protein_coding -13.02617318 0.003738147 ENSMUSG00000029319 'Coq2 protein_coding -13.02962541 0.003738197 ENSMUSG00000027367 'Stard7 protein_coding -13.03044832 0.003747572 ENSMUSG00000025733 'Rhot2 protein_coding -13.03323454 0.003739763 ENSMUSG00000027673 'Ndufb5 protein_coding -13.04007478 0.003736978 ENSMUSG00000042032 'Mat2b protein_coding -13.04042789 0.003737797 ENSMUSG00000003033 'Ap1m1 protein_coding -13.04123458 0.020488553 ENSMUSG00000021645 'Smn1 protein_coding -13.04205259 0.000198152 ENSMUSG00000031538 'Plat protein_coding -13.0494334 0.020491088 ENSMUSG00000108394 'RP24-333B11.4 TEC -13.07185045 0.004020813 ENSMUSG00000022148 'Fyb protein_coding -13.07511087 0.020346817 ENSMUSG00000027875 'Hmgcs2 protein_coding -13.09986539 0.00029714 ENSMUSG00000031834 'Pik3r2 protein_coding -13.10635699 0.000191356 ENSMUSG00000063065 'Mapk3 protein_coding -13.10817526 0.020314639 ENSMUSG00000025198 'Erlin1 protein_coding -13.11182148 0.003678077 ENSMUSG00000004931 'Apba3 protein_coding -13.1134332 0.000190392 ENSMUSG00000059182 'Skap2 protein_coding -13.11589335 0.003802627 ENSMUSG00000025591 'Tma16 protein_coding -13.11600195 0.003706521 ENSMUSG00000006024 'Napa protein_coding -13.13356628 0.020303759 ENSMUSG00000000149 'Gna12 protein_coding -13.13581762 0.020300479 ENSMUSG00000028339 'Col15a1 protein_coding -13.15082135 0.003691813 ENSMUSG00000032271 'Nnmt protein_coding -13.1545908 0.00020257 ENSMUSG00000029500 'Pgam5 protein_coding -13.15782842 0.003679627 ENSMUSG00000104528 'Gm43314 TEC -13.16079621 0.020254574 ENSMUSG00000042208 '0610010F05Rik protein_coding -13.17563376 0.003678183 ENSMUSG00000031762 'Mt2 protein_coding -13.19790215 0.02025472 ENSMUSG00000000581 'C1d protein_coding -13.20138355 0.003674493 ENSMUSG00000019872 'Smpdl3a protein_coding -13.20480975 0.020278493 ENSMUSG00000054203 'Ifi205 protein_coding -13.20634029 0.020266608 ENSMUSG00000025421 'Hdhd2 protein_coding -13.21368901 0.003786825 ENSMUSG00000060591 'Ifitm2 protein_coding -13.2176549 0.003681715 ENSMUSG00000021706 'Zfyve16 protein_coding -13.22331969 0.003681023 ENSMUSG00000033009 'Ogfod1 protein_coding -13.22683543 0.0001894 ENSMUSG00000024892 'Pcx protein_coding -13.23012868 0.003730343 ENSMUSG00000003099 'Ppp5c protein_coding -13.23221184 0.003661393 ENSMUSG00000042055 'Wdr11 protein_coding -13.23756811 0.003665175 ENSMUSG00000036160 'Surf6 protein_coding -13.23952263 0.000187057 ENSMUSG00000052406 'Rexo4 protein_coding -13.24211336 0.003663737 ENSMUSG00000021555 'Naa35 protein_coding -13.24715089 0.003666718 ENSMUSG00000030748 'Il4ra protein_coding -13.25299924 0.020273851 ENSMUSG00000092541 'Gm20537 protein_coding -13.25384938 0.004701666 ENSMUSG00000030204 'Ddx47 protein_coding -13.26007039 0.003667154 ENSMUSG00000037622 'Wdtc1 protein_coding -13.26170672 0.003667515 ENSMUSG00000071632 '2510002D24Rik protein_coding -13.26534823 0.003665567 ENSMUSG00000022521 'Crebbp protein_coding -13.26560603 0.003667353 ENSMUSG00000062014 'Gmfb protein_coding -13.26896482 0.020269878 ENSMUSG00000039678 'Tbc1d13 protein_coding -13.27190951 0.003676706 ENSMUSG00000015846 'Rxra protein_coding -13.27212553 0.020275831 ENSMUSG00000105742 'Gm42748 TEC -13.27864388 0.020303667 ENSMUSG00000022377 'Asap1 protein_coding -13.2916658 0.003664423 ENSMUSG00000015291 'Gdi1 protein_coding -13.29377637 0.003667556 ENSMUSG00000041164 'Zmiz2 protein_coding -13.29554599 0.003673314 ENSMUSG00000030002 'Dusp11 protein_coding -13.30134596 0.020272152 ENSMUSG00000042870 'Tom1 protein_coding -13.30135704 0.003690291 ENSMUSG00000106946 'Gm42856 TEC -13.30686242 0.003695836 ENSMUSG00000023030 'Slc11a2 protein_coding -13.32254933 0.020210274 ENSMUSG00000097042 'Gm17491 sense_intronic -13.32931607 0.020268379 ENSMUSG00000024799 'Tm7sf2 protein_coding -13.32976757 0.003673033 ENSMUSG00000004530 'Coro1c protein_coding -13.33013596 0.020275988 ENSMUSG00000024610 'Cd74 protein_coding -13.33364155 0.020249607 ENSMUSG00000026812 'Tsc1 protein_coding -13.35605162 0.003670563 Page 51 of 62 Diabetes
ENSMUSG00000029090 'Adgra3 protein_coding -13.35655774 0.003682602 ENSMUSG00000037300 'Ttc13 protein_coding -13.363836 0.003669037 ENSMUSG00000025533 'Asl protein_coding -13.36480965 0.00366886 ENSMUSG00000093262 'Gm23413 miRNA -13.36679632 0.020271439 ENSMUSG00000061353 'Cxcl12 protein_coding -13.36797525 0.003674139 ENSMUSG00000026174 'Cnot9 protein_coding -13.37551143 0.003671778 ENSMUSG00000031545 'Gpat4 protein_coding -13.38387621 0.020292874 ENSMUSG00000009646 'Pla2g12b protein_coding -13.40188939 0.020316483 ENSMUSG00000035898 'Uba6 protein_coding -13.40260665 0.003678522 ENSMUSG00000032691 'Nlrp3 protein_coding -13.4026895 0.020323519 ENSMUSG00000004929 'Thop1 protein_coding -13.40456108 0.003684589 ENSMUSG00000028896 'Rcc1 protein_coding -13.41180875 0.003675945 ENSMUSG00000021905 'Dph3 protein_coding -13.41765867 0.000186975 ENSMUSG00000028756 'Pink1 protein_coding -13.43073292 0.003689037 ENSMUSG00000021069 'Pygl protein_coding -13.43263446 0.020371686 ENSMUSG00000021364 'Elovl2 protein_coding -13.43592476 0.003699729 ENSMUSG00000030718 'Ppme1 protein_coding -13.45625054 0.000187908 ENSMUSG00000039934 'Gsap protein_coding -13.45842426 0.003714588 ENSMUSG00000032215 'Rsl24d1 protein_coding -13.46141621 0.003686578 ENSMUSG00000089809 'A930011G23Rik protein_coding -13.48638499 0.00042327 ENSMUSG00000040811 'Eml2 protein_coding -13.48927179 0.00018807 ENSMUSG00000022684 'Bfar protein_coding -13.49231406 0.003693186 ENSMUSG00000064215 'Ifi27 protein_coding -13.50281212 0.003771485 ENSMUSG00000104125 'Gm37488 TEC -13.50981163 0.02045127 ENSMUSG00000062963 'Ufc1 protein_coding -13.51752513 0.003715275 ENSMUSG00000019970 'Sgk1 protein_coding -13.52494472 0.004052912 ENSMUSG00000027879 'Sec22b protein_coding -13.53296595 0.003718401 ENSMUSG00000025702 'March8 protein_coding -13.5497974 0.020482617 ENSMUSG00000030654 'Arl6ip1 protein_coding -13.55017524 0.020487155 ENSMUSG00000018565 'Elp5 protein_coding -13.55135415 0.003716243 ENSMUSG00000078974 'Sec61g protein_coding -13.55883637 0.003717162 ENSMUSG00000034354 'Mtmr3 protein_coding -13.56795933 0.003746112 ENSMUSG00000045636 'Mtus1 protein_coding -13.57491097 0.003736756 ENSMUSG00000028527 'Ak4 protein_coding -13.57608325 0.000194546 ENSMUSG00000032217 'Rnf111 protein_coding -13.5897122 0.003719705 ENSMUSG00000054855 'Rnd1 protein_coding -13.59378615 0.003748105 ENSMUSG00000089929 'Bcl2a1b protein_coding -13.6036376 0.020524674 ENSMUSG00000105895 'Gm42829 sense_intronic -13.61059958 0.020487498 ENSMUSG00000004707 'Ly9 protein_coding -13.6180837 0.020478652 ENSMUSG00000032487 'Ptgs2 protein_coding -13.61963858 0.004557731 ENSMUSG00000009549 'Srp14 protein_coding -13.62305818 0.000189575 ENSMUSG00000027933 'Ints3 protein_coding -13.63080428 0.003726451 ENSMUSG00000028099 'Polr3c protein_coding -13.63304231 0.003721745 ENSMUSG00000028779 'Pef1 protein_coding -13.63680146 0.003731681 ENSMUSG00000030304 'Ergic2 protein_coding -13.63726411 0.003722659 ENSMUSG00000098557 'Kctd12 protein_coding -13.6375011 0.020547065 ENSMUSG00000022766 'Serpind1 protein_coding -13.65098744 0.003732553 ENSMUSG00000025907 'Rb1cc1 protein_coding -13.65164469 0.003726644 ENSMUSG00000031657 'Heatr3 protein_coding -13.65609832 0.003724665 ENSMUSG00000030225 'Dera protein_coding -13.67082363 0.003730807 ENSMUSG00000026245 'Farsb protein_coding -13.67105854 0.003728635 ENSMUSG00000018882 'Mrpl45 protein_coding -13.67811449 0.003721783 ENSMUSG00000014542 'Clec4f protein_coding -13.68273373 0.020536101 ENSMUSG00000040506 'Ambra1 protein_coding -13.68955951 0.003732809 ENSMUSG00000073775 'Kti12 protein_coding -13.70483864 0.003721974 ENSMUSG00000024571 'Gm16286 protein_coding -13.71561685 0.020535539 ENSMUSG00000032596 'Uba7 protein_coding -13.7161588 0.020533241 ENSMUSG00000024122 'Pdpk1 protein_coding -13.72246124 0.003716249 ENSMUSG00000024679 'Ms4a6d protein_coding -13.73138295 0.020572125 ENSMUSG00000078784 '1810022K09Rik protein_coding -13.76861008 0.00019002 ENSMUSG00000036733 'Rbm42 protein_coding -13.78396995 0.003711547 ENSMUSG00000026198 'Abcb6 protein_coding -13.83077135 0.003701717 ENSMUSG00000039221 'Rpl22l1 protein_coding -13.83135261 0.003679439 ENSMUSG00000042331 'Specc1 protein_coding -13.83542251 0.000189313 ENSMUSG00000000776 'Polr3d protein_coding -13.83680381 0.000186615 ENSMUSG00000022831 'Hcls1 protein_coding -13.88854309 0.004814057 ENSMUSG00000053565 'Eif3k protein_coding -13.90773039 0.003672962 ENSMUSG00000035370 'Adat3 protein_coding -13.91515877 0.003667724 ENSMUSG00000094806 'Cyp2d10 protein_coding -13.91934052 0.003684544 ENSMUSG00000038371 'Sbf2 protein_coding -13.92164511 0.003667439 ENSMUSG00000040479 'Dgkz protein_coding -13.93762655 0.003672851 ENSMUSG00000025580 'Eif4a3 protein_coding -13.97568184 0.003666252 ENSMUSG00000028670 'Lypla2 protein_coding -13.98217503 0.003656831 ENSMUSG00000025185 'Loxl4 protein_coding -13.98786588 0.003673237 ENSMUSG00000109005 'Gm45221 TEC -13.99627687 0.020370765 ENSMUSG00000029311 'Hsd17b11 protein_coding -14.00630719 0.000187863 ENSMUSG00000079293 'Clec7a polymorphic_pseudogene -14.00859202 0.020360181 ENSMUSG00000019370 'Calm3 protein_coding -14.03843985 0.003654857 ENSMUSG00000037373 'Ctbp1 protein_coding -14.06284359 0.003646778 ENSMUSG00000062590 'Armc9 protein_coding -14.10896939 0.020325932 ENSMUSG00000021540 'Smad5 protein_coding -14.11512066 0.000179966 ENSMUSG00000026917 'Wdr5 protein_coding -14.13190736 0.003621627 ENSMUSG00000028048 'Gba protein_coding -14.13251052 0.00363953 Diabetes Page 52 of 62
ENSMUSG00000036896 'C1qc protein_coding -14.14253412 0.020216235 ENSMUSG00000040025 'Ythdf2 protein_coding -14.15624807 0.000176945 ENSMUSG00000004788 'Eif2b2 protein_coding -14.17314464 0.000177061 ENSMUSG00000029190 'D5Ertd579e protein_coding -14.17596663 0.000463128 ENSMUSG00000004771 'Rab11a protein_coding -14.18666135 0.003585924 ENSMUSG00000055491 'Pprc1 protein_coding -14.19235689 0.003580971 ENSMUSG00000107215 'Gm43197 TEC -14.21592641 0.000183577 ENSMUSG00000018774 'Cd68 protein_coding -14.24717966 0.004060526 ENSMUSG00000052456 'Asna1 protein_coding -14.25247041 0.000172738 ENSMUSG00000000399 'Ndufa9 protein_coding -14.3065263 0.000169981 ENSMUSG00000048612 'Myof protein_coding -14.3497564 0.003787932 ENSMUSG00000041438 'Cirh1a protein_coding -14.37184812 0.003507709 ENSMUSG00000035325 'Sec31a protein_coding -14.38967889 0.019812305 ENSMUSG00000025044 'Msr1 protein_coding -14.42182395 0.004099561 ENSMUSG00000014402 'Tsg101 protein_coding -14.42641012 0.000164673 ENSMUSG00000030142 'Clec4e protein_coding -14.47122518 0.019515673 ENSMUSG00000069893 '9930111J21Rik1 protein_coding -14.48578589 0.019499507 ENSMUSG00000024712 'Rfk protein_coding -14.49258264 0.000159299 ENSMUSG00000032612 'Usp4 protein_coding -14.55374225 0.003351131 ENSMUSG00000022962 'Gart protein_coding -14.56197502 0.003345826 ENSMUSG00000030172 'Erc1 protein_coding -14.56213197 0.000155856 ENSMUSG00000085894 'Gm15832 processed_transcript -14.57453912 0.019217509 ENSMUSG00000017132 'Cyth1 protein_coding -14.60687754 0.003323159 ENSMUSG00000013629 'Cad protein_coding -14.62508526 0.003279436 ENSMUSG00000026395 'Ptprc protein_coding -14.64169094 0.019022513 ENSMUSG00000006273 'Atp6v1b2 protein_coding -14.64836508 0.003359383 ENSMUSG00000029545 'Acads protein_coding -14.66063859 0.000151108 ENSMUSG00000013787 'Ehmt2 protein_coding -14.7492576 0.000140237 ENSMUSG00000004849 'Ap1s1 protein_coding -14.76203888 0.000139035 ENSMUSG00000029192 'Tbc1d14 protein_coding -14.77232839 0.003151303 ENSMUSG00000030339 'Ltbr protein_coding -14.77603451 0.000137421 ENSMUSG00000062593 'Lilrb4a protein_coding -14.78791303 0.000229094 ENSMUSG00000042350 'Arel1 protein_coding -14.79614414 0.003194703 ENSMUSG00000015305 'Sash1 protein_coding -14.81150812 0.003297411 ENSMUSG00000059552 'Trp53 protein_coding -14.82704118 0.003062404 ENSMUSG00000032081 'Apoc3 protein_coding -14.85680237 0.018086723 ENSMUSG00000027282 'Mtch2 protein_coding -14.89753794 0.002970249 ENSMUSG00000058427 'Cxcl2 protein_coding -14.91066607 0.017832751 ENSMUSG00000029810 'Tmem176b protein_coding -14.93844328 0.002948936 ENSMUSG00000068749 'Psma5 protein_coding -15.05700441 0.000113309 ENSMUSG00000026965 'Anapc2 protein_coding -15.06028068 0.000113136 ENSMUSG00000032261 'Sh3bgrl2 protein_coding -15.13039121 0.00278605 ENSMUSG00000023224 'Serping1 protein_coding -15.18460196 0.002769423 ENSMUSG00000004730 'Adgre1 protein_coding -15.27930206 0.01642062 ENSMUSG00000034855 'Cxcl10 protein_coding -15.28416838 0.016406616 ENSMUSG00000029186 'Pi4k2b protein_coding -15.41339429 8.74E-05 ENSMUSG00000037902 'Sirpa protein_coding -15.48079949 0.003060768 ENSMUSG00000022967 'Ifnar1 protein_coding -15.5388339 7.59E-05 ENSMUSG00000002981 'Clptm1 protein_coding -15.64225113 6.60E-05 ENSMUSG00000002319 'Ipo4 protein_coding -15.86262926 5.48E-05 ENSMUSG00000020120 'Plek protein_coding -15.87999103 0.000181295 Page 53 of 62 Diabetes
Supplementary Table 2. Ingenuity Pathway Analysis (IPA) of up/down-regulated genes for canonical pathways from Supplementary Table 1 is shown.
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Probe Catalogue number (Applied Biosystems, Foster City, CA, USA) BIM Mm00437796_m1 (mouse) PPAR-γ Mm01208835_m1 (mouse) SCD1 Mm00772290_m1 (mouse) SREBP1 Mm00550338_m1 (mouse) ACC1 Mm01304277_m1 (mouse) CD36 MM00432403_m1 (mouse)
CD68 Mm00839636_g1
(mouse)
MCP-1 Mm00441242_m1
(mouse)
IL-6 Mm00446190_m1
(mouse)
18s Mm003928990_g1 (mouse)
Supplementary Table 3. List of probes used for qPCR. Real-time PCR was performed using the Rotor-Gene RG-3000 machine (Corbett Research; Qiagen, Hilden, Germany) and the TaqMan PCR Master Mix (AmpliTaq Gold with GeneAmp kit; Applied Biosystems) in 20µl reaction volumes.
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Antibody Company Reference Dilution BIM Cell Signaling, Danvers, MA #2933 1/1000 PUMA Cell Signaling, Danvers, MA #14570 1/1000 BCL-2 Cell Signaling, Danvers, MA #2870 1/1000 BCL-XL Cell Signaling, Danvers, MA #2764 1/1000 MCL-1 Cell Signaling, Danvers, MA #5453 1/1000 BAK Cell Signaling, Danvers, MA #3814 1/1000 BAX Cell Signaling, Danvers, MA #2772 1/1000 p-JNK Cell Signaling, Danvers, MA #9251 1/1000 JNK Cell Signaling, Danvers, MA #9252 1/1000 p-c-Jun Cell Signaling, Danvers, MA #9164 1/1000 c-Jun Cell Signaling, Danvers, MA #2315 1/1000 p-FoxO3a Cell Signaling, Danvers, MA #9466 1/1000 FoxO3a Cell Signaling, Danvers, MA #12829 1/1000 p-IR Cell Signaling, Danvers, MA #3024 1/1000 IR Cell Signaling, Danvers, MA #3025 1/1000 p-AKT Cell Signaling, Danvers, MA #4060 1/1000 AKT Cell Signaling, Danvers, MA #9272 1/1000 pSTAT1 BD Biosciences, CA 612132 1/1000 PTPox R&D Systems, Minneapolis, MN MAB2844 1/1000 p-Tyrosine Cell Signaling, Danvers, MA #9411 1/1000 p85 Merck Millipore, Billerica, MA ABS234 1/1000 IRS1 Merck Millipore, Billerica, MA 06-248 1/2000 β-actin Cell Signaling, Danvers, MA #3700 1/3000 α-tubulin SIGMA-ALDRICH, St Louis, MO #T9026 1/5000 HRP-conjugated anti- rabbit IgG Merck Millipore, Billerica, MA 12-348 1/5000 HRP-conjugated anti- mouse IgG DAKO, Denmark P0260 1/10000
Supplementary Table 4. List of antibodies used for Western blot analysis.
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Supplementary Figure 1. A: 10-week-old male C57BL/6 mice were fed with a high fat diet (18.4% protein, 23.5% fat, and 4.7% crude fibre) or maintained on a chow diet (20% protein, 6% fat, and 3.2% crude fibre) and body weights determined (n=10). B: Body composition (gonadal white adipose tissue (WAT), liver, and gastrocnemius muscle relative weights) was determined after chow or high fat feeding (n=9-10). C: Insulin tolerance tests (0.65mU insulin/g body weight; i.p.) were conducted in chow or high fat fed mice (n=10). D: Glucose tolerance tests (2mg glucose/g body weight; i.p.) were conducted in chow or high fat fed mice (n=10). E: Characteristics of the patients and liver biopsies preparations used for Western blot analysis (quantification shown in Fig. 1C). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.
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Supplementary Figure 2. A: Primary hepatocytes isolated from 8- to 12-week-old C57BL/6 mice were treated with 0.5mM palmitate at different time points, and processed for immunoblot analysis as indicated (n=3-4). B: Primary hepatocytes isolated from 8- to 12- week-old C57BL/6 mice were treated with 0.5mM oleate at different time points, and processed for immunoblot analysis as indicated (n=3). C: Liver extracts from chow versus 16- week high fat fed C57BL/6 male mice were processed and then analysed by immunoblotting as indicated (n=6). D: Primary hepatocytes isolated from 8- to 12-week-old C57BL/6 mice were treated with 0.5mM palmitate at different time points, and processed for immunoblot analysis as indicated (n=3).
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Supplementary Figure 3. A: Western blot showing specific deletion of BIM in the liver in BLKO mice (n=2). B: Mice were fed a standard chow diet and body weights recorded (n=10- 11). C: 10-week-old male lox/lox and BLKO mice were fed a chow diet for 25 weeks.
Oxygen consumption (VO2), energy expenditure, respiratory exchange ratio (RER=VCO2/VO2), daily food intake and ambulatory activity were assessed during the light and dark cycles for 2 consecutive days (n=7-10). *P < 0.05 vs. control.
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Supplementary Figure 4. A: High fat fed BLKO and control mice were injected with PBS or insulin (0.65mU insulin/g body weight, 10 min). Livers extracted were lysed in NP-40 buffer, and the lysates were precleared with Pansorbin (Calbiochem) for 30 min at 4°C. For IRS1 pull downs, 1µg of IRS1 antibody (Merck Millipore, #06-248) was bound to Protein A Agarose 4 Fast Flow (Sigma-Aldrich) for 3h at 4°C, followed by incubation with 500 µg of total liver lysate for 16h at 4°C under constant mixing. Immune complexes were washed four times with NP-40 buffer and once with 50mM Tris (pH 7.5), and then resolved by SDS-PAGE and immunoblotted with PI3K p85 (Merck Millipore, #ABS234) and IRS1 antibodies. B-C: 10- week-old BLKO and lox/lox male mice were high fat fed for 18 weeks, fasted for 6h and then injected with PBS or insulin (0.65mU/g body weight, 10 min). Muscle (B) and abdominal white adipose tissue (C) extracted and processed for immunoblotting with the indicated antibodies (n=3-7). D: 10-week-old male lox/lox and BLKO mice were fed a high fat diet for 16 weeks. Oxygen consumption (VO2), energy expenditure, respiratory exchange ratios (RER=VCO2/VO2), daily food intake and ambulatory activity were assessed during the light and dark cycles for 2 consecutive days (n=8). E: Representative fluorescence micrographs showing images of livers from 19-week high fat fed BLKO and lox/lox mice stained for TUNEL (red) and nuclear counterstained with DAPI (blue). Quantification of TUNEL positive cells/total cells per mouse is shown (n=4), more than 1000 cells were counted per
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mouse. White arrows show apoptotic cells. Bar, 250 µm. F: Livers were harvested from 4-h fasted mice, and processed for qPCR analysis of inflammatory markers as indicated (n=4-9). G: Serum levels of alanine aminotransferase (ALT) in obese BLKO (n=9) and lox/lox (n=9) mice. *P < 0.05; **P < 0.01 vs. control.
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Supplementary Figure 5. A: Western blot showing deficiency of BIM in the hepatocyte preparations used for RNA-seq analysis. B: 1,247 genes were significantly regulated by deficiency of BIM in 3 hepatocyte samples. Sequencing reads were mapped to the mouse genome version GRCm38/mm10 (http://www.ensembl.org). The reads were mapped with Tophat 2, program using mouse gene annotation from Ensembl version 86. An average of 93% of total reads were mapped. Gene quantification and abundance estimation from the mapped reads was performed with the FluxCapacitor program version 1.6.1. Lists of differentially expressed genes were generated using the FluxCapacitor program and edgeR analysis package. A gene was considered as expressed if it had a RPKM (‘reads per kilobase per million mapped reads’) greater than 0.5 and as “differentially expressed” as compared to lox/lox if it had a fold change higher that 1.5 (up-regulated) or lower than 0.6 (down-
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regulated) and a p-value of <0.05. The up/down-regulated genes were submitted to gene set enrichment analysis with Ingenuity Pathway Analysis (IPA). IPA of these up/down-regulated genes is shown for canonical pathways. The length of the blue bars indicates the significance of the association between the set of genes and the keyword, and is expressed as minus the logarithm of the probability that a random set of genes from the mouse genome would be associated with the same keyword. The cutoff on the p-value was 0.05 (corresponding to - log(p-value)=1.3 in the figure). For the full list of all significantly modified pathways and the genes affected genes see Supplementary Tables 1-2. C: Overview of mRNAs modified by BIM deficiency in hepatocytes. The figure summarizes the affected genes in the canonical pathways by IPA. D: Mitochondrial OCR was measured in BLKO and lox/lox hepatocytes using a Seahorse bioanalyzer. After recording three baseline OCR measurements, oligomycin (Oligo), FCCP, rotenone (Rot) and antimycin A (Anti) were sequentially added to the cells and OCR measurements taken thrice after each treatment. Quantification of parameters of mitochondrial respiration of triplicates is shown (n=2). E: Primary hepatocytes isolated from 8- to 12-week-old C57BL/6 mice were treated with 0.5mM palmitate at different time points, and processed for qPCR as indicated (n=2).